Therapeutic efficacy of Stephania tetrandra S. Moore for treatment of neovascularization of retinal capillary (retinopathy) in diabetes--in vitro study.

The present study was designed to examine therapeutic efficacy of the root extract of Stephania Tetrandra S. Moore (STMS) (traditional Chinese medicine; Han Fang Ji) for treatment of neovascularization of the retinal capillary (retinopathy) in streptozotocin (STZ)-induced diabetic rats (STZ diabetic rats) in culture. Recently we have established the culture system in which fetal bovine serum (FBS) in Dulbecco modified Eagle medium (DMEM) induced neovascularization of the retinal capillary and choroidal capillary in normal rats in culture. STZ diabetic rats showed more neovascularization of the retinal capillary and choroidal capillary than did normal rats in culture. In this study, the retinal tissue was removed for the posterior ocular region and cultured in DMEM containing FBS. The choroidal tissue of the posterior ocular region was also removed and cultured as an internal reference. Administration of STSM (0.91, 9.1 and 91 microg/ml) significantly suppressed neovascularization of the retinal capillary in both STZ diabetic rats and normal rats in a dose-dependent manner. Similar results were obtained with the choroidal capillary; administration of STSM suppressed neovascularization of the choroidal capillary in both STZ diabetic rats and normal rats. In order to determine the component of STSM inhibiting neovascularization of the retinal capillary, tetrandrine (a major chemical constituent of STSM) was administered and neovascularization of the retinal capillary was examined in culture. The effect of tetrandrine on the choroidal capillary was also examined as an internal reference. Administration of tetrandrine (0.1, 1.0 and 10 microM) suppressed neovascularization of the retinal capillary in both STZ diabetic rats and normal rats in a dose-dependent manner. Similar results were obtained with the choroidal capillary of both STZ diabetic rats and normal rats. We infer, therefore, that STSM has a direct effect on the retinal capillary of posterior ocular region and suppresses neovascularization of retinal capillary in STZ diabetic rats through the activation of tetrandrine. These results suggest that STSM may prevent for delay the progression of retinopathy in diabetic patients.     

Enhancement of tissue plasminogen activator production from human normal fibroblast IMR-90 cells by limitation of cell growth

Summary Tissue plasminogen activator (t-PA) production induced by proteose peptone from IMR-90 cells was investigated. Cells monolayered on plastic surfaces had a higher ability to produce t-PA per unit cell compared to those grown tri-dimensionally on ceramic pieces. Furthermore, confluent monolayers of the cells, which suffered contact inhibition and resulted in limited growth, were available for t-PA production. Repeated batch production with microcarriers, on which the cells were almost confluent monolayers similar to those in T-flasks, was performed. Utilization of the cells, which had limited serum in the growth phase, resulted in an increase in production. Moreover, dilution of the basal components of the medium at initiation of the production phase markedly promoted t-PA production. The volumetric productivity was stable for 30 days at 100 IU/cm³ per day. The cells were then mostly retained on microcarriers. Thus, an effective and scalable method of t-PA production by normal fibroblast cells was developed. Offprint requests to: S. Mitsuda     

An inquiry into the selective protection of glycine during the radiolysis of glycine-alanine mixtures in aqueous solutions and its implications to the preservation of optically active amino acids in the early earth

Akaboshi et al. (1990) has found an unexpected protection of the achiral amino acid, glycine, towards ionizing radiation at the expense of the selective destruction of the chiral amino acids, alanine and aspartic acid. The present work examines the mechanism of this protection for the case of alanine. We have developed a computer model for the radiolysis of glycine, alanine and glycine-alanine mixtures in aqueous solution. It is established that this protection is due in part to the reaction of the α-radical of glycine with alanine to regenerate a more stable α-radical, according to the following reaction, $$ \cdot CH(NH_3^ + )CO_2^ - + CH_3 CH(NH_3^ + )CO_2^ - \to CH_2 (NH_3^ + )CO_2^ - + CH_3 \dot C(NH_3^ + )CO_2^ -$$ The rate constant of this reaction was estimated to be ≤10⁴M^-1s^-1. The implications for this selective protection of glycine are considered for a hypothetical case in which there would be an enrichment of about 10% ofL-alanine in the primitive ocean and taking the glycine/alanine ratios obtained in CH₄-and CO₂- dominated atmospheres using electric discharge experiments. It is predicted that alanine would be rapidly destroyed and radioracemized in spite of the fact that the concentration of alanine is equal or significantly lower than that of glycine. Assuming that chiral amino acids were a prerequisite for the origin of life, it can be deduced that life could have appeared in a relatively short period of time unless there was a constant supply of optical amino acids from extraterrestrial sources.     

Protein synthesis induced by infection with packaged lambda dv plasmid

Plasmid λdv or imm²¹dv DNA was joined to a λ arm having a cos site. This recombinant plasmid can be packaged in a λ head, and used to infect Escherichia coli K12 cells. The injected DNA molecules become plasmids in cells. By adding these particles to uv-irradiated uvrA cells, the packageable λdv or imm²¹dv plasmids can be induced to synthesize proteins coded by genes on the plasmid genome. The packageable plasmid system is thus suitable for studying on synthesis and regulation of plasmid-coded biopolymers. Analyses of the dv-coded proteins in gel electrophoreses revealed that among several genes carried on the dv plasmid genome, only those genes that are members of the pRoR-tof-cII-O-P operon can be expressed. Evidence has been presented to show that expression of this operon, which is directly correlated with replication of the genome, is only partially allowed in cells perpetuating the dv plasmid. These observations are discussed in connection with the autorepressor model (D. E. Berg, 1974, Virology62, 224–233; K. Matsubara, 1976, J. Mol. Biol.102, 427–439) that genetically accounts for the control mechanism of plasmid replication.     

Study on the chemical evolution of low molecular weight compounds in a highly oxidized atmosphere using electric discharges

The molecular basis for the chemical evolution of low molecular compounds was studied using electric discharges on a higly oxidized atmosphere comprised of CO₂, N₂ and H₂O. In the gas phase, O₂ and CO were formed by the decomposition of CO₂ and their yields were enhanced by the addition of N₂ to the gas mixture. It was demonstrated that H₂O suppressed the reduction of CO₂ while H₂O also had a role in producing organic compounds such as formic acid and formaldehyde. Infrared analysis of the water soluble products and the inner surface of the reaction vessel indicated the production of compounds more complex than formic acid and formaldehyde. These compounds contained the chemical bonds which were identified to be OH, CO, CN and/or CC.     

Formation of amino acids from reactor-irradiated ammonium acetate

Ammonium acetate in various conditions was irradiated in a reactor to examine the contributions of both the reactor radiations and recoiled¹⁴C nucleis to form the biologically interesting molecules. Present investigations demonstrated that several amino acids, glycine, alanine, -alanine and GABA, and may-be aspartic acid, serine and valine by prolonged irradiation, were formed in the aqueous solutions of ammonium acetate.¹⁴C-radioactivities were also found distributed in these amino acids. However, no special relationship between¹⁴C-radioactivity and these amino acids formed was observed.     

Asymmetrical radical formation in D- and L-alanines irradiated with yttrium-90β-rays

Radical formation in^{9 0}Y--irradiated D- and L-alanines was studied using ESR. It was observed that the relative radical concentration by -irradiation was distinguishably (13.9–21.5%) more in D-alanine than in L-alanine. Discussion was made on the possible mechanisms for the observed results.