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.     

Synthesis of 3,6-di-O-Methylglucosyl Disaccharides with Methyl 3-(p-Hydroxyphenyl)propionate as a Linker Arm and Their Use in the Serodiagnosis of Leprosy

The disaccharide, 2,3-di-O-methyl-4-O-(3,6-di-O-methyl-β-d-glucopyranosyl)-l-rhamno-pyranose, the distal segment of phenolic glycolipid I, that is a specific antigen from Mycobacterium leprae, and some related disaccharides were synthesised as the glycosides of methyl 3-(p-hydroxyphenyl)propionate. The methyl 3-(p-hydroxyphenyl)propionate was coupled with 2,3,4-tri-O-acetyl-l-rhamnosyl bromide, deacetylated, acetonated, coupled with 2,4,6-tri-O-acetyl-3-O-methyl-d-glucosyl bromide, and converted into a variety of p-(2-methoxycarbonylethyl)phenyl 4-O-(3,6-di-O-methyl-d-glucopyranosyl)-containing disaccharides that are amenable to ready conjugation with protein carriers, thereby providing neo-glycoconjugates for the specific serodiagnosis of leprosy.     

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.     

Biochemical Studies on the Mineral Components in Sake Yeast

The critical concentrations of minerals in a growing medium for maximum fermentation of yeast were as follows: P, 1 mmol/1; Mg, 0.2 mmol/1; and K, 1~2 mmol/1. These values are lower than those for the saturation of the cells with each mineral. The order of the concentration for maximum fermentation (K>P>Mg) is in agreement with that for yeast growth.

Only a small amount of mineral salt was required to increase the fermentative activity. Very small increase of fermentative activity was observed when the starved yeast was enriched with corresponding minerals by incubating cells with the mineral salt and glucose.     

Studies on Vitamin B6 Metabolism in Microorganisms

Escherichia freundii alkaline phosphatase was found in a membrane fraction and was purified by procedures involving spheroplast formation with lysozyme and EDTA, and DEAE-cellulose and Sephadex G-150 column chromatographies. Then this enzyme along with other phosphatases was investigated on the ability to transfer the phosphoryl group from p-nitrophenyl phosphate to pyridoxine. It was found that the ability of the transphosphorylation varied with these phosphatases. The transphosphorylation to hydroxy compounds such as alcohols, sugars and nucleosides was also compared. Escherichia freundii acid phosphatase showed the highest activity of transphosphorylation among phosphatases tested. The mechanism of transphosphorylation was discussed.

An enzyme, pyridoxamine 5′-phosphate transaminase, was purified from the cell-free extract of Clostridium kainantoi. The purification procedures involved ammonium sulfate fractionation, protamine sulfate treatment and, DEAE-cellulose, hydroxylapatite, DEAE-Sephadex and Sephadex G-200 column chromatographies. The purified enzyme, which had approximately 2700-fold higher specific activity over the original extract, showed a single schlieren pattern in the ultracentrifuge. From the spectral analysis, it seemed that pyridoxamine 5′-phosphate transaminase did not contain pyridoxal 5′-phosphate as a prosthetic group. It was recognized that the transamination was accelerated by the addition of amino acid and was inhibited by diisopropyl phosphofluoride. Glutamic acid formed in the reaction was identified to be a D-isomer. A study on the substrate specificity showed that the enzyme might be possible to be specific for pyridoxamine 5′-phosphate.

The extracellular formation of vitamin B₆ was searched in marine and terrestrial microorganisms. Two bacterial strains were selected and were identified as Vibrio and Flavobacterium, respectively. Marine microorganisms showed the considerable formation of vitamin B₆ and the presence of vitamin B₆ in sea water was also recognized. The cultural and reaction conditions for vitamin B₆ formation by these strains were investigated. Glycerol was commonly the most effective compound on vitamin B₆ formation among the compounds tested. It was suggested that both bacteria did not have the control system on vitamin B₆ biosynthesis by the amount of possible end products.     

Localization of 9- and 13-oxo-octadecadienoic acids in tomato fruit

We previously reported that the two peroxisome proliferator-activated receptor-α agonists, 9- and 13-oxo-octadecadienoic acids (oxo-ODAs), were found in the tomato fruit. However, their localization remains unknown. Herein, we showed that oxo-ODAs localize primarily in the fruit peel and their amount increases after the homogenization of the tomato fruit.     

Generation of the antioxidant yellow pigment derived from 4-methylthio-3-butenyl isothiocyanate in salted radish roots (takuan-zuke)

2-[3-(2-Thioxopyrrolidin-3-ylidene)methyl]-tryptophan (TPMT) is a yellow pigment of salted radish roots (takuan-zuke) derived from 4-methylthio-3-butenyl isothiocyanate (MTBITC), the pungent component of radish roots. Here, we prepared salted radish and analyzed the behavior of the yellow pigment and related substances in the dehydration process and long-term salting process. All salted radish samples turned yellow, and their b^* values increased with time and temperature. The salted radish that was sun-dried and pickled at room temperature turned the brightest yellow, and the generation of TPMT was clearly confirmed. These results indicate that tissue shrinkage due to dehydration, salting temperature, and pH play important roles in the yellowing of takuan-zuke.     

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.     

Individualized Mutation Detection in Circulating Tumor DNA for Monitoring Colorectal Tumor Burden Using a Cancer-Associated Gene Sequencing Panel

Background

Circulating tumor DNA (ctDNA) carries information on tumor burden. However, the mutation spectrum is different among tumors. This study was designed to examine the utility of ctDNA for monitoring tumor burden based on an individual mutation profile.

Methodology

DNA was extracted from a total of 176 samples, including pre- and post-operational plasma, primary tumors, and peripheral blood mononuclear cells (PBMC), from 44 individuals with colorectal tumor who underwent curative resection of colorectal tumors, as well as nine healthy individuals. Using a panel of 50 cancer-associated genes, tumor-unique mutations were identified by comparing the single nucleotide variants (SNVs) from tumors and PBMCs with an Ion PGM sequencer. A group of the tumor-unique mutations from individual tumors were designated as individual marker mutations (MMs) to trace tumor burden by ctDNA using droplet digital PCR (ddPCR). From these experiments, three major objectives were assessed: (a) Tumor-unique mutations; (b) mutation spectrum of a tumor; and (c) changes in allele frequency of the MMs in ctDNA after curative resection of the tumor.

Results

A total of 128 gene point mutations were identified in 27 colorectal tumors. Twenty-six genes were mutated in at least 1 sample, while 14 genes were found to be mutated in only 1 sample, respectively. An average of 2.7 genes were mutated per tumor. Subsequently, 24 MMs were selected from SNVs for tumor burden monitoring. Among the MMs found by ddPCR with > 0.1% variant allele frequency in plasma DNA, 100% (8 out of 8) exhibited a decrease in post-operation ctDNA, whereas none of the 16 MMs found by ddPCR with < 0.1% variant allele frequency in plasma DNA showed a decrease.

Conclusions

This panel of 50 cancer-associated genes appeared to be sufficient to identify individual, tumor-unique, mutated ctDNA markers in cancer patients. The MMs showed the clinical utility in monitoring curatively-treated colorectal tumor burden if the allele frequency of MMs in plasma DNA is above 0.1%.