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.     

Development of the alt Mutant of Pisum sativum L

The alt (albina-terminalis) mutant of Pisum sativum L. germinates normally, produces several nodes, and then above a sharp transition produces 2 to 3 bleached nodes, ceases growth, and eventually dies. Green nodes have normal chlorophyll content, absorption spectra, photosynthetic rates, and ultrastructure. In bleaching tissues, the chloroplasts degenerate rapidly, followed by extensive disruption and loss of the remaining cytoplasm and organelles. Application of tissue extracts of normal genotypes of pea, corn, and bean stimulates apical development of alt. The resulting tissues have essentially normal structure and function. Application of thiamine, thiamine monophosphate, and thiamine pyrophosphate also stimulate normal apical development at concentrations of 1 micromolar and above. Partial characterization of the stimulus from pea seed extracts is consistent with thiamine as the active factor.     

五种雀形目鸟类核型的比较研究

染色体核型的比较研究,对于了解物种的特性,探索物种的遗传、进化、系统发育及分类地位都有一定的意义。有关鸟类的核型研究,国外已有不少报道(Castrovicja,1969; Hammar,1966,1975; Ray-Chaudhuri et al.,1969;Shidds,1982;Ta—kagi,1972等)国内在这方面的工作开展较迟,已作过核型分析的鸟类为数不多,仅见王应祥等(1982)的报道。迄今,作过核型研究的鸟类已达500多种,其中雀形目有200种。本文比较分析了五种野生雀形目鸟类的核型,它们是黑头蜡嘴雀,斑鸫,黄腹山雀,红尾伯劳和灰背椋鸟。     

Protein translocation into Escherichia coli membrane vesicles is inhibited by functional synthetic signal peptides

A synthetic peptide corresponding to the signal sequence of wild type Escherichia coli lambda-receptor protein (LamB) inhibits in vitro translocation of precursors of both alkaline phosphatase and outer membrane protein A into E. coli membrane vesicles (half-maximal inhibition at 1-2 microM). By contrast, the inhibitory effect was nearly absent in a synthetic peptide corresponding to the signal sequence from a mutant strain that harbors a deletion mutation in the LamB signal region and displays an export-defective phenotype for this protein in vivo. Two peptides derived from pseudorevertant strains that arose from the deletion mutant and exported LamB in vivo were found to inhibit in vitro translocation with effectiveness that correlated with their in vivo export ability. Controls indicated that these synthetic signal peptides did not disrupt the E. coli membrane vesicles. These results can be interpreted to indicate that the presequences of exported proteins interact specifically with a receptor either in the E. coli inner membrane or in the cytoplasmic fraction. However, biophysical data for the family of signal peptides studied here reveal that they will spontaneously insert into a lipid membrane at concentrations comparable to those that cause inhibition. Hence, an indirect effect mediated by the lipid bilayer of the membrane must be considered.     

Synergism between eosinophil cationic protein and oxygen metabolites in killing of schistosomula of Schistosoma mansoni

To study the cytotoxic reactions responsible for mediating eosinophil damage to schistosomula of Schistosoma mansoni, we have used cytoplasts (eosinophil or neutrophil vesicles devoid of granules and nuclei, with an intact oxidase in their plasma membrane) in combination with purified eosinophil cationic protein (ECP) or major basic protein (MBP) in a cytotoxicity test toward schistosomula. Suboptimal concentrations of ECP (10(-6) M) or MBP (10(-6) M) resulting in less than 10% killing were used in combination with cytoplasts. Cytoplasts alone in the presence of immune serum tested over a wide range of cytoplast:schistosomula ratios generated superoxide and hydrogen peroxide, but were unable to damage schistosomula. However, when a suboptimal ECP concentration (10(-6) M) was combined with neutroplasts or eosinoplasts, 43.9% +/- 8.5 (n = 7) and 24.7% +/- 9.8 (n = 3), respectively, of the schistosomula were killed. Oxygen metabolites were responsible for the synergism, because cytoplasts from a patient with chronic granulomatous disease were unable to act in synergy with ECP. In contrast to ECP, no synergism was found between cytoplasts and MBP (10(-6) to 2 X 10(-5)M). These results show that oxygen metabolites are important for the killing of schistosomula by lowering the concentration of ECP needed to inflict damage.     

Prohead and DNA-gp3-dependent ATPase activity of the DNA packaging protein gp16 of bacteriophage phi 29

The ATPase activity of the DNA packaging protein gp16 (gene product 16) of bacteriophage phi 29 was studied in the completely defined in-vitro assembly system. ATP was hydrolyzed to ADP and Pi in the packaging reaction that included purified proheads, DNA-gp3 and gp16. Approximately one molecule of ATP was used in the packaging of 2 base-pairs of phi 29 DNA, or 9 X 10(3) ATP molecules per virion. The hydrolysis of ATP by gp16 was both prohead and DNA-gp3 dependent. gp16 contained both the "A-type" and the "B-type" ATP-binding consensus sequences (Walker et al., 1982) and the predicted secondary structure for ATP binding. The A-type sequence of gp16 was "basic-hydrophobic region-G-X2-G-X-G-K-S-X7-hydrophobic", and similar sequences were found in the phage DNA packaging proteins gpA of lambda, gp19 of T7 and gp17 of T4. Having both the ATP-binding and potential magnesium-binding domains, all of these proteins probably function as ATPases and may have common prohead-binding capabilities. The phi 29 protein gp3, covalently bound to the DNA, may be analogous in function to proteins gpNul of lambda and gpl of phi 21 that bind the DNA.     

Stimulations of arachidonate release and inositol-1,4,5-triphosphate formation are mediated by distinct G-proteins in human platelets

Addition of fluoroaluminate to human platelet suspension stimulated thromboxane synthesis and inositol-1,4,5-triphosphate formation in a time and dose dependent manner. Neomycin inhibited markedly fluoroaluminate induced inositol-1,4,5-triphosphate formation without significantly affecting thromboxane synthesis. Preincubation of platelets with PGE1, also inhibited significantly inositol-1,4,5-triphosphate formation with modest reduction of thromboxane synthesis. On the contrary, pretreatment of platelets with pertussis toxin inhibited fluoroaluminate stimulated thromboxane synthesis without affecting inositol-1,4,5-triphosphate formation. Similarly, preincubation of platelets with phorbol ester, PMA, inhibited markedly thromboxane synthesis with modest reduction of inositol-1,4,5-triphosphate formation. These results indicate that inositol-1,4,5-triphosphate formation and arachidonate release and thromboxane synthesis are controlled separately and are mediated by different G-proteins which are coupled to phospholipase C and phospholipase A2 respectively in platelets.     

Effects of some alcohols on the conformation of mitochondrial H+-ATPase complex and F1-ATPase from pig heart

The conformations of the H+-ATPase complex and F1-ATPase in low concentrations of methanol, ethanol, n-propanol, iso-propanol and t-butanol were studied by circular dichroism. For F1-ATPase, all but methanol first increased and then decreased the circular dichroism magnitude of helical bands as the alcohol concentration was increased. With ethanol, n-propanol, iso-propanol and t-butanol, the alpha-helix content reached a maximum at about 5% alcohol and began to decrease at 10%. The content of beta-sheet showed the opposite effect, reaching a minimum at 5% and increasing slightly at higher concentrations. None of the alcohols studied had a significant effect on the conformation of the H+-ATPase complex. This difference implies that the alcohols had a greater effect on free F1-ATPase than on the membrane-bound F1-ATPase. The hydrophobic protein F0 and the membrane lipids in the H+-ATPase complex may stabilize and protect F1 from the effects of the alcohols.     

Kinetic basis for insensitivity to tetrodotoxin and saxitoxin in sodium channels of canine heart and denervated rat skeletal muscle

The single-channel blocking kinetics of tetrodotoxin (TTX), saxitoxin (STX), and several STX derivatives were measured for various Na-channel subtypes incorporated into planar lipid bilayers in the presence of batrachotoxin. The subtypes studied include Na channels from rat skeletal muscle and rat brain, which have high affinity for TTX/STX, and Na channels from denervated rat skeletal muscle and canine heart, which have about 20-60-fold lower affinity for these toxins at 22 degrees C. The equilibrium dissociation constant of toxin binding is an exponential function of voltage (e-fold per 40 mV) in the range of -60 to +60 mV. This voltage dependence is similar for all channel subtypes and toxins, indicating that this property is a conserved feature of channel function for batrachotoxin-activated channels. The decrease in binding affinity for TTX and STX in low-affinity subtypes is due to a 3-9-fold decrease in the association rate constant and a 4-8-fold increase in the dissociation rate constant. For a series of STX derivatives, the association rate constant for toxin binding is approximately an exponential function of net toxin charge in membranes of neutral lipids, implying that there is a negative surface potential due to fixed negative charges in the vicinity of the toxin receptor. The magnitude of this surface potential (-35 to -43 mV at 0.2 M NaCl) is similar for both high- and low-affinity subtypes, suggesting that the lower association rate of toxin binding to toxin-insensitive subtypes is not due to decreased surface charge but rather to a slower protein conformational step. The increased rates of toxin dissociation from insensitive subtypes can be attributed to the loss of a few specific bonding interactions in the binding site such as loss of a hydrogen bond with the N-1 hydroxyl group of neosaxitoxin, which contributes about 1 kcal/mol of intrinsic binding energy.