A homologous sequence between H+-ATPase (F0F1) and cation-transporting ATPases. Thr-285----Asp replacement in the beta subunit of Escherichia coli F1 changes its catalytic properties

A sequence of 10 amino acids (I-C-S-D-K-T-G-T-L-T) of ion motive ATPases such as Na+/K+-ATPase is similar to the sequence of the beta subunit of H+-ATPases, including that of Escherichia coli (I-T-S-T-K-T-G-S-I-T) (residues 282-291). The Asp (D) residue phosphorylated in ion motive ATPase corresponds to Thr (T) of the beta subunit. This substitution may be reasonable because there is no phosphoenzyme intermediate in the catalytic cycle of F1-ATPase. We replaced Thr-285 of the beta subunit by an Asp residue by in vitro mutagenesis and reconstituted the alpha beta gamma complex from the mutant (or wild-type) beta and wild-type alpha and gamma subunits. The uni- and multisite ATPase activities of the alpha beta gamma complex with mutant beta subunits were about 20 and 30% of those with the wild-type subunit. The rate of ATP binding (k1) of the mutant complex under uni-site conditions was about 10-fold less than that of the wild-type complex. These results suggest that Thr-285, or the region in its vicinity, is essential for normal catalysis of the H+-ATPase. The mutant complex could not form a phosphoenzyme under the conditions where the H+/K+-ATPase is phosphorylated, suggesting that another residue(s) may also be involved in formation of the intermediate in ion motive ATPase. The wild-type alpha beta gamma complex had slightly different kinetic properties from the wild-type F1, possibly because it did not contain the epsilon subunit.     

Purification and identification of [hydroxyprolyl3]bradykinin in ascitic fluid from a patient with gastric cancer

Kinins in the ascitic fluid from a patient with gastric cancer were purified by gel filtration and reversed-phase high-performance liquid chromatography (HPLC). Two fractions (fractions I and II) showed kinin activity. Fraction I did not correspond to either bradykinin or other known kinins, whereas fraction II corresponded to bradykinin. Fraction I contained 8 amino acid residues from bradykinin minus 1 proline plus 1 additional hydroxyproline. Sequence analysis of fraction I showed that the proline at the third amino acid residue of bradykinin was replaced by hydroxyproline. The retention time of fraction I on reversed-phase HPLC was exactly the same as that of synthetic [hydroxyprolyl3]bradykinin (Arg-Pro-Hyp-Gly-Phe-Ser-Pro-Phe-Arg) and was distinguishable from des-Pro3-bradykinin. Thus, these results demonstrate for the first time the presence of [hydroxyprolyl3]bradykinin in vivo. This is also the first report of the presence of bradykinin in human tumor ascites.     

Growth experiment of Hantaan virus in A549 cells: an attempt to improve the immunofluorescent antibody technique for hemorrhagic fever with renal syndrome

An assay method for the infectivity of Hantaan virus, a causative agent of HFRS (hemorrhagic fever with renal syndrome), was developed by the use of IFA (immunofluorescent antibody technique). With the aid of this method, the growth characteristics of Hantaan virus, 76-118 strain, were followed in A549 cells. At a maximal MOI (multiplicity of infection) of 1.6 VAIU (viral antigen-inducing units) per cell, the conventionally available value, plateau level potencies of the viral antigen and virus infectivity were attained at eight and ten days postinfection, respectively, and most of the infective virus produced accumulated in the culture fluids of infected cells. When infections were defined with MOI values in terms of VAIU per cell, development of the viral antigen was highly consistent and followed a given pattern of kinetics. Based on these findings, a protocol for preparation of the viral antigen in IFA was presented, wherein spot culture and FBS treatment were emphasized as effective procedures to minimize non-specific staining.     

Recessive Nonsense Suppressors in SACCHAROMYCES CEREVISIAE : Action Spectra, Complementation Groups and Map Positions

Three genes SUP111, SUP112 and SUP113 of Saccharomyces cerevisiae have been identified that can mutate to give recessive omnipotent nonsense suppressors. Alleles of these loci can also act as allosuppressors; that is, different phenotypes, due apparently to different efficiencies of suppression, can result from different alleles at a given locus. The SUP111, SUP112 and SUP113 loci map to the right arms of chromosomes VIII, VII and XIII, respectively.     

Interleukin 1 alpha mRNA in virus-transformed T and B cells

IL-1 alpha cDNA clone was isolated from a T cell line infected by the human T lymphotropic retrovirus type-I (HTLV-I/ATLV). We found significant amounts of mRNA hybridizing to IL-1 alpha cDNA not only in HTLV-I-transformed T cells but also in Epstein-Barr Virus-transformed B cells. A part of IL-2 receptor inducing activity in Adult T cell leukemia (ATL) cell line seems to be due to IL-1 alpha.     

Genetic studies on the muscle protein turnover rate of coturnix quail

The validation of the urinary excretion of N-methylhistidine (N-MH) by quail as an index of the muscle protein turnover rate was tested using the criterion of the rate of recovery of radioactivity in urine following an intraperitoneal dose of l-[3-¹⁴C]methylhistidine. A genetic study on muscle protein turnover in quail was conducted using three genetically diverse lines (LL, large body size; SS, small body size; RR, random-bred control line) selected for body size. When l-[3-¹⁴C]methylhistidine was administered to 20-week-old male and female coturnix quail by direct intraperitoneal injection, approximately 90% of the l-[3-¹⁴C]methylhistidine was recovered by 96 hr postinjection. Recoveries were low in the egg and muscle. These results show that N-MH released from myofibrillar protein is not reutilized and the excretion of N-MH is a satisfactory index of muscle protein breakdown. In all lines, the amount of urinary N-MH excretion and fractional synthesis (K_s) and degradation (K_d) rates at the high growing period were higher than those at the low growing period. The K_s and K_d are significantly different among selected lines at both 3 and 6 weeks of age. At 3 weeks of age, the fractional rate of synthesis of the LL line (13.2%/day) was higher than that of the RR line (11.5%/day), whereas the SS (8.1%/day) was lower than that of the RR line (11.5%/day). The fractional rates of degradation of both the LL line (4.1%/day) and the SS line (5.6%/day) were lower than that of the RR line (7.0%/day) at 3 weeks of age. From these results, it was recognized that selection for body size gave rise to the changes in the muscle protein turnover rate.     

Mutagenic activation of aflatoxin B1 by several forms of purified cytochrome P-450

Metabolic activation by several forms of purified cytochrome P-450 of aflatoxin B1 to a product(s) mutagenic to Salmonella typhimurium TA100 was examined. Of the 5 forms of cytochrome P-450 purified from liver microsomes of untreated and PCB-treated male rats, a constitutive form purified from untreated male rats, P-450-male, and a high-spin form of cytochrome P-450, P-448-H, from PCB-treated rats were highly active.     

Effects of phenobarbital, 3-methylcholanthrene and polychlorinated biphenyls on sex-specific forms of cytochrome P-450 in liver microsomes of rats

The effects of treatment with phenobarbital, 3-methylcholanthrene or polychlorinated biphenyls (PCB) on the amounts of sex-specific forms of cytochrome P-450, namely P-450-male and P-450-female, in male and female rats were studied. Although treatment with phenobarbital, 3-methylcholanthrene or PCB markedly increased the total amount of hepatic cytochrome P-450, P-450-male and P-450-female were rather decreased or not significantly changed. Thus, the percentages of P-450-male and P-450-female in the total cytochrome P-450 were decreased in liver microsomes from the treated rats. The increases in specific cytochrome P-450, such as P-448-H, P-448-L, and P-450I-c accounted for the increase in the total amount of cytochrome P-450 in the treated rats. The treatment with phenobarbital or PCB increased the activities of testosterone 16 alpha-hydroxylase, benzo(a)pyrene hydroxylase and aminopyrine N-demethylase more markedly in female rats than in male rats. Similarly, the treatment with 3-methylcholanthrene increased benzo(a)pyrene hydroxylase more markedly in female rats. Therefore, the sex-differences in testosterone 16 alpha-hydroxylase, benzo(a)pyrene hydroxylase, and aminopyrine N-demethylase activities became smaller after the drug treatment. These results indicate that sex-specific P-450-male and P-450-female were unaffected, or even depressed by the agents in some cases.