Bioproduction of prostaglandins in a transgenic liverwort, Marchantia polymorpha

Prostaglandins are biologically active substances used in a wide range of medical treatments. Prostaglandins have been supplied mainly by chemical synthesis; nevertheless, the high cost of prostaglandin production remains a factor. To lower the cost of prostaglandin production, we attempted to produce prostaglandins using a liverwort, Marchantia polymorpha L., which accumulates arachidonic acid, which is known as a substrate of prostaglandins. Here we report the first bioproduction of prostaglandins in plant species by introducing a cyclooxygenase gene from a red alga, Gracilaria vermiculophylla into the liverwort. The transgenic liverworts accumulated prostaglandin F_2α, prostaglandin E₂ and prostaglandin D₂ which were not detected in the wild-type liverwort. Moreover, we succeeded in drastically increasing the bioproduction of prostaglandins using an in vitro reaction system with the extracts of transgenic liverworts.     

Studies on the Phenylphenol Derivatives with Biological Activity

More than ninety phenylphenol derivatives were tested for fungistatic activity toward ten species of agriculturally important fungi. One of N-methylcarbamates and some nitro- or chloro-substituted derivatives were highly toxic against Piricularia oryzae. From the relationship between the chemical structure and the activity, it is supposed that the reactivity, the permeability, the steric effect and the metabolic activation of the compound are the responsible factors for the fungistatic activity.     

Effect of Dietary Composition on Lipids in Serum and in Liver of Rats Fed a Cystine-excess Diet

The effects of dietary composition on lipids in serum and in liver of rats fed with a cystine- excess diet were investigated.

When starch was used as the carbohydrate source, the addition of excess-cystine caused an increase in serum cholesterol and phospholipids, and hepatomegaly. Phospholipids in serum of rats fed with a cystine-excess diet containing 5% corn oil were higher than those with a cystine-excess diet that was low in corn oil (0.1 %). The addition of konjac mannan and pectin prevented hypercholesterolemia, and the rise in phospholipids in serum was prevented by the addition of konjac mannan.

Liver cholesterol (mg/liver/100 g of body wt.) increased in rats fed with a cystine-excess diet.

The addition of excess cystine to a diet containing sucrose as the carbohydrate source resulted in a marked increase of cholesterol in serum and liver, and a decrease of serum triglycerides.

The replacement of starch by sucrose in the cystine-excess diet increased liver cholesterol.

Lipids, cholesterol, phospholipids and triglycerides in the liver, but not phospholipids, when expressed as mg per g of liver for rats fed with the diets containing sucrose, increased when compared to those for rats fed with the diets containing starch. In contrast, serum triglycerides increased.     

Colorimetric Determination of 2-Chlorophenyl N-Methylcarbamate

2-Chlorophenyl N-methylcarbamate was determined colorimetrically. It was hydrolyzed with a veronal buffer solution (pH 8.0) to give corresponding 2-chlorophenol, which was coupled 4-nitrobenzenediazonium fluoroborate to produce a color having a maximum absorption at 520 mμ. The developed color was very stable. On the other hand, contaminated 2-chlorophenol was adsorbed in treated alumina before being hydrolyzed. Other contaminated impurities gave little influences. Technical materials and some formulated products were determined and good results were obtained.     

Screening for Antioxidants of Microbial Origin

Asymmetric hydrolysis of (dl)-1-acyloxy-2-halo-1-phenylethanes by lipoprotein lipase Amano P from Pseudomonas fluorescens and the lipase from Chromobacterium viscosum afforded the optically active (R) residual substrates and (S)-2-halo-1-hydroxy-1-phenylethanes in 100% enantiomeric excess (e.e.). The length of acyl residues from acetyl to octanoyl in the substrates did not influence the enantioselectivity.

Both enantiomers of optically active styrene oxides were synthesized from the enzymatic products.     

Change of the Gluconeogenic Capacity of Rat Kidney Cortex Slices with Dietary Components

Substituting protein for carbohydrate in diets significantly enhanced the rates of glucose formation from pyruvate, glutamate, or glycerol in rat kidney cortex slices. The tissue, however, increased slightly its gluconeogenic capacity in response to low carbohydrate, high fat diet. The rates of glucose taken up per unit weight of kidney cortex of rats fed a high carbohydrate diet was higher than those of rats fed diets high in protein or fat. Kidney weight in g per 100 g body weight (relative kidney size) of rats fed diets high in protein was significantly higher than that of rats fed diets high in carbohydrate or fat.     

Impact of Diffusion Barriers to Small Cytotoxic Molecules on the Efficacy of Immunotherapy in Breast Cancer

Molecular-focused cancer therapies, e.g., molecularly targeted therapy and immunotherapy, so far demonstrate only limited efficacy in cancer patients. We hypothesize that underestimating the role of biophysical factors that impact the delivery of drugs or cytotoxic cells to the target sites (for associated preferential cytotoxicity or cell signaling modulation) may be responsible for the poor clinical outcome. Therefore, instead of focusing exclusively on the investigation of molecular mechanisms in cancer cells, convection-diffusion of cytotoxic molecules and migration of cancer-killing cells within tumor tissue should be taken into account to improve therapeutic effectiveness. To test this hypothesis, we have developed a mathematical model of the interstitial diffusion and uptake of small cytotoxic molecules secreted by T-cells, which is capable of predicting breast cancer growth inhibition as measured both in vitro and in vivo. Our analysis shows that diffusion barriers of cytotoxic molecules conspire with γδ T-cell scarcity in tissue to limit the inhibitory effects of γδ T-cells on cancer cells. This may increase the necessary ratios of γδ T-cells to cancer cells within tissue to unrealistic values for having an intended therapeutic effect, and decrease the effectiveness of the immunotherapeutic treatment.     

A soluble form of human nectin-2 impairs exocrine secretion of pancreas and formation of zymogen granules in transgenic mice

Transgenic mouse lines expressing a soluble form of human nectin-2 (hNectin-2Ig Tg) exhibited distinctive elevation of amylase and lipase levels in the sera. In this study, we aimed to clarify the histopathology and to propose the transgenic mouse lines as new animal model for characteristic pancreatic exocrine defects. The significant increase of amylase and lipase levels in sera of the transgenic lines approximately peaked at 8 weeks old and thereafter, plateaued or gradually decreased. The histopathology in transgenic acinar cells was characterized by intracytoplasmic accumulation of abnormal proteins with decrease of normal zymogen granules. The hNectin-2Ig expression was observed in the cytoplasm of pancreatic acinar cells, which was consistent with zymogen granules. However, signals of hNectin-2Ig were very weak in the transgenic acinar cells with the abnormal cytoplasmic accumulaion. The PCNA-positive cells increased in the transgenic pancreas, which suggested the affected acinar cells were regenerated. Acinar cells of hNectin-2Ig Tg had markedly small number of zymogen granules with remarkable dilation of the endoplasmic reticulum (ER) lumen containing abundant abnormal proteins. In conclusion, hNectin-2Ig Tg is proposed as a new animal model for characteristic pancreatic exocrine defects, which are due to the ER stress induced by expression of mutated cell adhesion molecule that is a soluble form of human nectin-2.