Purification and characterization of two serine isoacceptor tRNAs from bovine mitochondria by using a hybridization assay method.

For large scale preparation of mitochondrial tRNAs, a new hybridization assay method using synthetic oligodeoxyribonucleotide probes (16-17mer) complementary to individual tRNA sequences was developed and applied for the purification of two serine isoacceptor tRNAs (tRNASerAGY and tRNASerUCN) from bovine mitochondria. It is about 100 times more sensitive than the conventional aminoacylation assay method. 2-4 A260 units each of both tRNASer isoacceptors were purified from 17.5 kg of bovine liver, and they were characterized by means of nuclease digestion, melting profiles and aminoacylation activity. It is suggested that tRNASerUCN possesses the D loop/T loop interaction like usual L-shaped tRNAs, and that tRNASerAGY lacking almost an entire D arm is aminoacylated with an efficiency not very much lower than that of tRNASerUCN.     

Adaptive on-line optimizing control of bioreactor systems

Several on-line optimizing control strategies were proposed and tested by computer simulation for the efficient operation of bioreactors. The control task was divided into two, one of which was to search for the optimal operating point and passed the set point to the lower layer of which task was to make the process output follow the set point as soon as possible. It was shown to be effective for the upper layer to express the objective function as a polynomial with respect to the measurement variable and to make use of it for finding the optimum point. Noting that the major dynamic characteristics of bioreactor system is the time-varying and nonlinear nature, the adaptive type control system is in evitable. It was shown to be quite effective to use discrete type self-tuning PID controller and the optimal controller compensated for the interaction between the control loops.Application was made to the cell recycle system for the production of lactic acid and baker's yeast cultivation. I was found from the former application that the control quality can be significantly improved by incorporating the decoupling strategy into the lower layer closed-loop system. It was also found from the latter application that the initial startup period can be significantly reduced by making use of the rough mathematical model.     

Evidence for chromosomal replicons as units of sister chromatid exchanges

Chromosomal replicons have been described as the cytological counterpart of DNA replicon clusters and have previously been studied in vitro using premature chromosome condensation-sister chromatid differentiation (PCC-SCD) techniques. Chromosomal replicons are visualized as small SCD segments in S-phase cells, and measurement of these segments can provide estimates of relative chromosomal replicon size corresponding to DNA replicon clusters functioning coordinately in S-phase. Current hypotheses of sister chromatid exchange (SCE) formation postulate that sites of SCE induction are associated with active replicons or replicon clusters. We have applied the PCC-SCD technique to in vivo studies of mouse bone marrow cells that have been treated with cyclophosphamide (CP) for two cell cycles. We have been able to visualize chromosomal replicons, as well as SCEs which have been induced in vivo by CP treatment, simultaneously in the same cells. Chromosomal replicons visualized as small SCD segments were measured in PCC cells classified at early or late S-phase based on SCD segment size prevalence. Early S-phase (E/S) PCC cells contained 90% of the SCD segments measured clustered in a segment size range of 0.1 to 0.8 m with a peak value around 0.3 to 0.6 m regardless of CP treatment. As the cells progressed through S-phase, late S-phase (L/S) PCC cells were characterized by the appearance of larger SCD segments and even whole SCD chromosomes in addition to small SCD segments. A concentration of units around 0.4 to 1.0 m was found for L/S SCD segment size distributions regardless of CP treatment with an apparent bimodal profile. Our in vivo data support the existence of a subunit organization of chromosomal replication with a basic functional unit being 0.3 to 0.6 m in size. In addition, we have found that this chromosomal unit of replication or chromosomal replicon does not seem to be functionally perturbed by the mutagen CP. We also found that small SCD segments of 0.4 to 0.7 m in length were involved in the formation of an SCE, suggesting that both spontaneous and CP-induced SCEs occur between chromosomal replicons. These findings provide direct cytogenetic evidence to support a replicon cluster/chromosomal replicon model for SCE formation.     

Nippostrongylus brasiliensis: radioresistant IgE antibody-forming cells in infected rats

In Nippostrongylus brasiliensis-infected rats, anti-N. brasiliensis IgE antibody production was observed at 20 weeks postinfection, long after the worms, as a source of antigen, had been expelled. The persistent IgE production was not abrogated after whole body irradiation (800 R) administered at 12 or 20 weeks, suggesting the participation of radioresistant IgE-forming cells. Help of T cells and recruitment of B memory cells in the irradiated rats seems to be ruled out by the findings that the irradiation completely inhibited the initiation of anti-N. brasiliensis IgE production in rats shortly after the infection with N. brasiliensis or after primary and secondary immunization with N. brasiliensis-antigen. Moreover, clearance of anti-N. brasiliensis IgE antibody from circulation did not seem to be crucially affected by the irradiation. The radioresistant cells forming anti-N. brasiliensis IgE were most productive in mesenteric lymph nodes as compared to other lymph nodes. The recognition of antigens fractionated by chromatography on Sephadex G-200 was the same for IgE-forming cells from rats 12 weeks after infection as for those from 3 weeks after infection. Based on these results, one of the mechanisms of persistent elevation of IgE antibody in the host infected with helminth parasites might be explained by the participation of radioresistant IgE-forming cells.     

Synthesis of human endothelin-1 precursors in Escherichia coli

Endothelin, the most potent vasoconstrictor found in nature, is thought to be important in the regulation of blood pressure and/or local blood distribution. Human placenta cDNA fragment encoding preproendothelin-1 (preproET-1) and its carboxyl terminal mature precursor (C-matured precursor) was expressed in E. coli. These products were characterized by both enzyme immunoassay and Western blot analysis.     

Amino acid sequence and relative biological activity of eel atrial natriuretic peptide

A peptide exhibiting vasodepressor and natriuretic activities in rats was isolated from eel atria, and its primary structure was determined as H-Ser-Lys-Ser-Ser-Ser-Pro-Cys-Phe-Gly-Gly-Lys-Leu-Asp-Arg-Ile-Gly-Ser-Tyr-Ser- Gly-Leu-Gly-Cys-Asn-Ser-Arg-Lys-OH. This peptide, termed eel atrial natriuretic peptide (ANP), has sequence homology of 59% to mammalian (human or rat) ANP, 52% to fowl ANP, and 46% to frog ANP. When the biological activity of synthetic eel ANP was compared with that of human ANP, the eel peptide was 110 times more potent for the vasodepressor activity in eels, nearly equipotent for the vasodepressor activity in quails, and 20 times less potent for the vasodepressor and natriuretic activity in rats.     

Two distinct types of endothelin receptors are present on chick cardiac membranes

Competitive displacement experiments of 125I-endothelin (ET)-1, -2, or -3 binding to chick cardiac membranes were performed with unlabeled ET-1, -2, -3, and sarafotoxin S6b (STX) as competitors. 125I-ET-1 and -2 binding was competitively inhibited by increasing concentrations of these unlabeled peptides in the same order; i.e. ET-2 greater than or equal to ET-1 greater than ET-3 greater than STX. In contrast, the order of potency in displacing 125I-ET-3 binding was ET-3 greater than ET-2 greater than or equal to ET-1 greater than STX. Affinity labeling of the membranes by cross-linking with 125I-ET-1 and -2 via disuccinimidyl tartarate yielded one major specific band with an apparent Mr = 53,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by autoradiography. On the other hand, affinity labeling with 125I-ET-3 showed that two major and one minor bands of Mr = 34,000, 46,000, and 53,000, respectively, were specifically labeled. These results indicate the presence of two distinct types of ET receptors, one of which has higher affinity for ET-1 and -2 than ET-3 and the other is conversely ET-3-preferring.     

Induction of manganese superoxide dismutase by tumor necrosis factor in human breast cancer MCF-7 cell line and its TNF-resistant variant

Tumor necrosis factor (TNF)-resistant variant of human mammary cancer MCF-7 cell line was isolated by stepwise selection. The final TNF-resistant variant Tnf-1000 showed more than 100-fold higher resistance than the parental MCF-7 cell. Saturation kinetics for 125I-TNF binding showed that TNF-1000 cells had similar TNF receptor numbers as MCF-7 cells, but of a lower affinity. Induction of superoxide dismutase (SOD) was compared between MCF-7 and Tnf-1000 cells treated with TNF: SOD scavenges potentially toxic superoxide radicals. TNF induced more mitochondrial manganese SOD (SODm) in MCF-7 than in Tnf-1000 whereas there appeared to be no significant induction of cytosolic copper/zinc SOD (SODc) by TNF in both MCF-7 and Tnf-1000 cell lines. Acquirement of TNF-resistance in MCF-7 cells might be correlated with expression of SODm.