Detection of Trp-P-1 and Trp-P-2, carcinogenic tryptophan pyrolysis products, in dialysis fluid of patients with uremia

In order to estimate the exposure levels of mutagenic and carcinogenic heterocyclic amines in humans, we developed a high-performance liquid chromatography method to detect 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) in dialysis fluid of patients with uremia. Using this methods, dialysis fluid of 12 patients who had received hemodialysis treatment or continuous ambulatory peritoneal dialysis was examined. Trp-P-1 was detected in dialysate of all uremic patients (727 +/- 282 pmoles, n = 12). In patients who had been treated with continuous ambulatory peritoneal dialysis, the average amount of Trp-P-1 found in whole dialysate (6 l) per day was 710 +/- 203 pmoles (mean +/- S.D., n = 8). Moreover, Trp-P-2 could be detected in 5 out of 12 patients (206 +/- 85 pmoles, n = 5). These results indicate that patients with uremia are actually exposed to carcinogenic tryptophan pyrolysis products. The average exposure level of Trp-P-1 in uremic patients apparently exceeded 710 pmoles (150 ng) per day.     

Protein Synthesis in Bromegrass (Bromus inermis Leyss) Cultured Cells during the Induction of Frost Tolerance by Abscisic Acid or Low Temperature

Bromus inermis Leyss cell cultures treated with 75 micromolar abscisic acid (ABA) at both 23 and 3°C developed more freezing resistance than cells cultured at 3°C. Protein synthesis in cells induced to become freezing tolerant by ABA and low temperature was monitored by [¹⁴C]leucine incorporation. Protein synthesis continued at 3°C, but net cell growth was stopped. Most of the major proteins detected at 23°C were synthesized at 3°C. However, some proteins were synthesized only at low temperatures, whereas others were inhibited. ABA showed similar effects on protein synthesis at both 23 and 3°C. Comparative electrophoretic analysis of [¹⁴C]leucine labeled protein detected the synthesis of 19, 21 and 47 kilodalton proteins in less than 8 hours after exposure to exogenous ABA. Proteins in the 20 kilodalton range were also synthesized at 3°C. In addition, a 31 kilodalton protein band showed increased expression in freezing resistant ABA treated cultures after 36 hours growth at both 3 and 23°C. Quantitative analysis of [¹⁴C]leucine labeled polypeptides in two-dimensional gels confirmed the increased expression of the 31 kilodalton protein. Two-dimensional analysis also resolved a 72 kilodalton protein enriched in ABA treated cultures and identified three proteins (24.5, 47, and 48 kilodaltons) induced by low temperature growth.     

Characterization of neutral glycosphingolipids from fetal human brain: evidence for stage-specific expression of the globo, ganglio, and neolacto series in the central nervous system

Neutral glycosphingolipids were isolated from normal human fetal brains, at 22 to 23 weeks gestation. They were identified as monohexosylceramides, lactosylceramide, and glycolipids belonging to the globo (globotriaosylceramide) and ganglio (gangliotriaosylceramide) series. In addition, considerable amounts of neolactotetraosylceramide and III3-alpha-fucosyl-neolactotetraosylceramide were detected. Although neutral glycolipids of the globo, ganglio, and neolacto series have been demonstrated in the brains of cases with some sphingolipidoses, they are not present in appreciable amounts in differentiated normal brain. Therefore, the present and previous observations would imply that the metabolism of these glycolipid series actively occurs in the normal brain at an early stage of differentiation and continues thereafter in the brain in the case of some sphingolipidoses. The diseased brain is most probably accompanied by a disturbance of differentiation.     

Induction of alkaline phosphatase in primary cultures of epiphyseal growth plate chondrocytes by a serum-derived factor

Alkaline phosphatase (AP) activity in epiphyseal growth plate cartilage increases markedly during differentiation of the chondrocytes, and reaches high levels in the zone of hypertrophy where vascular penetration and provisional mineralization begin. A proteinaceous factor has been discovered in serum that stimulates the expression of AP in chicken growth plate chondrocytes when these cells are grown in serum-free media. Sera from a variety of vertebrate species (goat, fetal bovine, horse, human, and chicken) all contained detectable levels of the inducing activity. The chondrocyte AP-induction factor (CAP-IF) from fetal bovine serum was precipitated with ammonium sulfate between 33% and 50% saturation, and purified by dye-ligand affinity chromatography. The active fraction, which eluted from an Affi-Gel Blue column between 0.10 and 0.15 M NaCl, was further resolved on a QMA anion exchange column. The most active and almost homogeneous fraction contained primarily a 64.5 kDa protein; about 3 micrograms/ml medium induced 50% of the maximal level of AP induction. CAP-IF is stable to heat (100 degrees C for 3 min) and dithiothreitol (50 mM) treatment, and is only mildly inactivated by 2 h treatment with trypsin. CAP-IF caused no significant effect on cell division as measured by 3H-thymidine uptake. Time-course studies revealed that at least 18-24 h exposure of the chondrocytes to CAP-IF is required to produce major increases in AP activity. Longer exposure time generally further increases the response. Cycloheximide almost completely blocked the increase in AP activity, indicating that de novo protein synthesis is required for induction.     

Occurrence of ceramide-glycanase in the earthworm, Lumbricus terrestris

We have detected the presence of ceramide-glycanase in the earthworm, Lumbricus terrestris. We have also devised a simple method for the preparation of this enzyme from the earthworm. This enzyme cleaved the linkage between the ceramide and the glycan chain in LacCer, GbOse3Cer, GbOse4Cer, GbOse5Cer, GM3, GM2, GM1 and GD1a. By using tritium-labeled GM2 as substrate, the optimum pH of this enzyme was found to be between pH 4 and 4.5. In the earthworm, the ceramide-glycanase was mainly found in the muscle. The intestine was found to contain a very low level of this enzyme. Because of their easy availability, earthworms should become a convenient source for the preparation of ceramide-glycanase.     

Initial-rate studies of a thermophilic glucokinase from Bacillus stearothermophilus.

The initial rates of phosphorylation of glucose catalysed by glucokinase from Bacillus stearothermophilus were measured over a wide range of glucose, MgATP2-, MgADP- and glucose 6-phosphate concentrations. The results of the effects of the inhibitors on the initial rates suggest that the reaction mechanism is essentially the ordered Bi Bi, in which glucose adds to the enzyme before MgATP2- and glucose 6-phosphate is released from the enzyme after the dissociation of MgADP-, and also suggest that the final step in which glucose 6-phosphate is released is irreversible. For many reaction schemes, the rate equations were derived on the basis of the pseudo-steady-state assumption and were used to correlate the experimental rate data. From this result, we concluded that the reaction obeys the ordered mechanism accompanied by the formation of a non-productive ternary complex, glucose-MgADP--enzyme. By using the experimental Dalziel coefficients phi i, some kinetic parameters were evaluated. The enzyme was characterized by the thermal stability and the low Michaelis constant, the values of which were 54 microM for glucose and 32 microM for MgATP2-.     

Sister-chromatid exchanges in lymphocytes of workers exposed to trichloroethylene

To detect mutagenic effects of trichloroethylene (TCE) on humans, sister-chromatid exchanges (SCEs) were analyzed in lymphocytes of 22 workers occupationally exposed to TCE and 22 matched controls. Although urinalysis in the workers revealed their obvious exposure to TCE, no increase in SCE frequencies was found in lymphocytes of the workers. SCE analysis in lymphocytes could not detect mutagenic effects by occupational exposure to TCE on humans.     

Electron Transfer between Plastocyanin and P700 in Various Types of Photosystem I Complex

Three types of PS I Chl-protein complex, PS I 180, PS I 65,and PS I 30, have been prepared and the kinetic properties ofthe transfer of electrons from plastocyanin to P700 in the PSI complexes with different sized antennae were examined. ThePS I 180 complex, which consists of 180 Chi per P700, showedthe almost same rate constant and effects of cations for thetransfer of electrons from plastocyanin to P700 as those obtainedwith PS I-enriched membrane fragments. The rate constant increasedwith the addition of low concentrations of monovalent and divalentcations, but decreased with high concentrations of cations.However, the rate was severely reduced in the case of the PSI 65 and PS I 30 complexes, and quite different effects of cationswere observed. Given the presence of additional 25- to 28-kDapolypeptides in the PS I 180 complex as compared to the PS I65 and PS I 30 complexes, we discuss a possible function forthese polypeptides in the regulation of the reaction betweenplastocyanin and P700. ¹This work was supported in part by a Grant-in-Aid for ScientificResearch from the Ministry of Education, Science and Cultureof Japan. (Received May 27, 1988; Accepted November 7, 1988)