Utilization of Amino Acids as a Sole Source of Nitrogen by Obligate Chemolithoautotroph Thiobacillus ferrooxidans

An obligate chemolithoautotroph, Thiobacillus ferrooxidans API 9–3, could utilize amino acids, other than glycine, methionine and phenylalanine, as a sole source of nitrogen. However, both the growth rate and growth yield were lower than those in Fe²⁺-NH4 -salts medium, suggesting that the ammonium ion was a superior nitrogen source for the strain compared to amino acids. Methionine and phenylalanine strongly inhibited the cell growth on Fe²⁺-NH4-salts medium at 10 mm. [¹⁴C]Glycine could not be taken up into the cells, and this meant the strain could not use glycine as a sole source of nitrogen. The uptake of [¹⁴C]leucine into the cells was dependent on the presence of Fe^{2 +}. When the strain was cultured on Fe^{2 +} - leucine (lOmm)-salts medium lacking an inorganic nitrogen source for 5 days at 30°C, 83.5% and 16.5% of the cellular carbon were derived from carbon dioxide and leucine, respectively, indicating that carbon dioxide was a superior carbon source for the bacterium compared to leucine. The ammonium ion did not inhibit the utilization of leucine for cellular carbon. Leucine uptake was markedly inhibited by inhibitors of protein synthesis, such as chloramphenicol (94.3% at 1 mm), streptomycin (57.2% at 5mm) and rifampin (77.2% at 0.1 mm), respectively. Carbon dioxide uptake was also completely inhibited by chloramphenicol at 4mm. These results suggest that the transport of both amino acids and carbon dioxide into the cells was dependent on protein synthesis.     

The Effect of Riboflavin on Hypertension induced by High Salt Diet in the Rat

The effect of riboflavin on development of hypertension in rats given a high salt diet was studied. Large doses of riboflavin prevented both elevation of blood pressure and rise of cholesterol levels in the serum. The increase in liver monoamine oxidase activity of the rats fed riboflavin was confirmed.     

Effect of Heavy Metal Ions on the Growth and Iron-oxidizing Activity of Thiobacillus ferrooxidans

Effect of heavy metal ions on the growth and the iron-oxidizing activity of Thiobacillus ferrooxidans were investigated.

Cupric, zinc, cadmium, and chromium ions had no effect on the growth and the iron-oxidizing activity of cell suspensions or cell-free extracts of the bacterium in high concentrations (10^?3~10^?2M). Lead ion delayed the start of the growth slightly in 10^?3 M, but it did not inhibit the iron-oxidizing activity of the cells in the concentration. Tin and molybdenum oxide ions inhibited both of them in the concentration above 10^?3 M.

Mercuric mercurous, and silver ions had the most harmful effect. In the concentration of 10^?3 .M, each of the cations inhibited almost completely both the growth and the iron-oxidizing activity of the cells.

In the experiments with cell-free extracts it was observed that the activity of cytochrome oxidase (cytochrome a₅₉₇) operating in the iron-oxidizing system of the bacterium was specifically inhibited with mercuric ion in the concentration above 5 × 10^?4 M.     

Influence of Forced-Feeding of Individual Essential Amino Acids on the Activities of All Urea Cycle Enzymes in Rat Liver

The response of all urea cycle enzymes, i.e. carbamyl phosphate synthetase, ornithine transcarbamylase, argininosuccinate synthetase, argininosuccinase and arginase, has been determined in the liver of protein-depleted young rats which were forcibly fed individual essential l-amino acids along with or without caloric sources. The feeding of individual amino acids produced different effects on the level of each of the enzymes, and generally the response of carbamyl phosphate synthetase, argininosuccinate synthetase, argininosuccinase and arginase was greater than that of ornithine transcarbamylase. Of all the essential amino acids tested tryptophan was most effective on the elevation of these enzymes. Several amino acids, phenylalanine, leucine, threonine and methionine had also somewhat effect on the increase of some enzyme activities, but other amino acids had little or no effect on the response of these enzymes. On the contrary, histidine and lysine caused appreciable decrease of arginase activity. These enzyme activities in rats fed tryptophan alone were extremely higher than those of animals fed it along with caloric sources. The response level of the enzymes was essentially dependent on the tryptophan content in diets under the proper conditions. Tryptophan feeding did not produce any increase in both levels of urine and plasma urea despite the elevation of all urea cycle enzyme activities occured.     

Identification of the Female Sex Pheromone of the Cabbage Webworm

Gibberellins A₄ and A₃₆ were identified from flowering and vegetative apices of ten month-old sugarcane (Saccharum spp. hybrids) plants. The identifications were based on retention times, relative to authentic standards, on sequential silica gel partition column chromatography→bioassay→C₁₈ reverse phase high performance liquid chromatography→bioassay→ capillary gas chromatography (GC), and on GC-selected ion monitoring (SIM), the relative intensities of six characteristic ions being monitored in comparison with authentic standards.     

Acid-stable α-Amylase of Black Aspergilli

Some general properties of the acid-stable dextrinizing amylase of black Aspergillus were investigated comparing with those of Taka-amylase A. The mode of action on starch of this amylase was quite similar to that of Taka-amylase A. Saccharifying degree at red point in starch-iodine color reaction was 5.1% and the limit of starch saccharification was a little over 40 per cent calculated as glucose with both amylases. Maltase activity was absent. Degradation products in the course of starch hydrolysis were also quite similar and they mutarotated downward. So this amylase was decided to be α-type. Thermal stability of the acid-stable α-amylase was higher than that of Taka-amylase A. Its acid stability was much higher than that of Taka-amylase A. Taka-amylase A was inactivated completely at pH 2.2, 37°C, for 30 min, but the acid-stable α-amylase retained 87% of its original activity.

From the amylase preparation of black Aspergillus acid-stable α-amylase and acidunstable α-amylase were separated by gel filtration on sephadex G-100 column. From the acid-unstable α-amylase fraction this enzyme was purified by fractionations with rivanol and acetone, and finally obtained as a homogeneous protein after gel filtration with sephadex G-50. Comparison of some general properties between the two α-amylases was carried out. Catalytic action was quite similar with both enzymes, but dextrinizing unit per mg enzyme protein of the acid-unstable α-amylase was about 5.6 times as large as that of the acid-stable α-amylase. The acid-unstable α-amylase was less heat-stable than the acid-stable α-amylase. Acid stability and pH-activity curve were compared with both α-amylases. High stability of the acid-stable α-amylase in acidic condition was observed, but, in alkaline range, it was more sensitive than the acid-unstable α-amylase.     

Studies on Erythorbic Acid Production by Fermentation

Screening was carried out for erythorbic acid (EA)-producing strains from about 5,000 newly isolated fungi and bacteria. Penicillium notatum FY 115 was screened out as most powerful EA producer. Only Penicillium, but no other genera, was obtained as EA producers from our screening program. Monospore selections and mutagenic treatments succeeded to elevate the yield of EA over 40% to glucose supplied. Various cultural conditions were studied, and pH change during fermentation process was proved to be most important for favorable EA production. Over 80% yield could be obtained when washed mycelium was used in dilute glucose solution.

Abundant accumulation of EA by the strain FY 115, Penicillium sp., in fermentation broth was studied, and EA, both free and Na-salt, was obtained as crystal in the yield of about 45% to glucose supplied, in the media of 8% glucose by jar fermentor, in considering the inhibitory effect of some metal ion.

Extraction processes were improved to elevate the yield and was developed the continuous multi-bed extraction system of anion-exchange resin, which resulted in the yield of 90.9% of EA from fermentation broth in sum total.     

Physiological Studies on Thiobacilli

The electrophoretically pure preparation of cytochrome c from Thiobacillus thiooxidans was obtained. The absorption spectrum exhibited maxima at 415, 521 and 550 mμ in reduced form. The various properties of the cytochrome were very close to these of mammalian cytochrome c, i. e., absorption spectrum, electrophoretic pattern, isoelectric point and E₀′.

Electrophoretically homogenous preparation of NADPH-cytochrome c oxidoreductase was isolated from the soluble fraction of Thiobacillus thiooxidans. The purification of the enzyme was carried out using the fractionation with ammonium sulfate, the treatment with Amberlite IRC-50 and the disk electrophoresis.     

Inhibition of the Growth Factor MDK/Midkine by a Novel Small Molecule Compound to Treat Non-Small Cell Lung Cancer

Midkine (MDK) is a heparin-binding growth factor that is highly expressed in many malignant tumors, including lung cancers. MDK activates the PI3K pathway and induces anti-apoptotic activity, in turn enhancing the survival of tumors. Therefore, the inhibition of MDK is considered a potential strategy for cancer therapy. In the present study, we demonstrate a novel small molecule compound (iMDK) that targets MDK. iMDK inhibited the cell growth of MDK-positive H441 lung adenocarcinoma cells that harbor an oncogenic KRAS mutation and H520 squamous cell lung cancer cells, both of which are types of untreatable lung cancer. However, iMDK did not reduce the cell viability of MDK-negative A549 lung adenocarcinoma cells or normal human lung fibroblast (NHLF) cells indicating its specificity. iMDK suppressed the endogenous expression of MDK but not that of other growth factors such as PTN or VEGF. iMDK suppressed the growth of H441 cells by inhibiting the PI3K pathway and inducing apoptosis. Systemic administration of iMDK significantly inhibited tumor growth in a xenograft mouse model in vivo. Inhibition of MDK with iMDK provides a potential therapeutic approach for the treatment of lung cancers that are driven by MDK.