Ehime University Institutional Repository

Ehime University (愛媛大学)博士(農学)thesi

Md., Shafiqul Islam

（第３号様式）(Form No. 3) 
学  位  論  文  要  旨  
Dissertation Abstract 
 
 
氏名：  
Name 
Md. Shafiqul Islam 
 
学位論文題目： 
Title of Dissertation 
Allelopathic Potential and Allelochemicals in Four Weed 
Species  
(4種の雑草種のアレロパシー活性とアレロパシー物質 )  
学位論文要旨：  
Dissertation Abstract 
 
 
 
 
Allelopathy is an important interference mechanism between donor and receptor plants in 
the environment. In allelopathy, allelochemicals are released from plants which have an 
effect on the growth, survival and development of its associated plants. The allelopathic 
potential of plants and allelochemicals are suggested as an alternative tool of managing 
weeds. During the search for plants with allelopathic potential, there has been a lot of focus 
on discovering allelopathic weed species. During this search, many species of weed have 
been studied and have been discovered to have allelopathic properties.  
Four different species of weed, namely Fimbristylis dichotoma  L. (Cyperaceae), Cyperus 
difformis L. (Cyperaceae), Marsilea crenata  Presl. (Marsileaceae) and Rumex maritimus  L. 
(Polygonaceae) were used in the study to investigate their allelopathic potential and to 
search allelochemicals. The aqueous methanol extracts of F. dichotoma , C. difformis, M. 
crenata and R. maritimus showed significant inhibitory effects on the shoot and root growth 
of cress, lettuce, alfalfa, rapeseed, barnyard grass, Italian ryegrass, timothy and foxtail 
fescue test plants in a way which was concentration -dependent. Total inhibition of shoot 
and root growth of cress, lettuce, alfalfa, and foxtail fescue was discovered at 0.3 g DW 
equivalent extract/mL by the extracts of C. difformis , F. dichotoma , M. crenata , and R. 
maritimus. Different inhibition levels were also discovered by the extracts when the tested 
plants underwent other concentrations of treatment.  The I50 values (concentration required 
for 50% inhibition) of four weed species for shoot and root growth of all test plants also 
differed. Concentration- and species- specific growth inhibitory effects of F. dichotoma , C. 
difformis, M. crenata and R. maritimus on the test plants suggest that these weeds have 
allelopathic potential and therefore might possess allelochemicals with allelopathic 
properties.  
（第３号様式）(Form No. 3) 
Two growth inhibitory substances were isolated after a series of chromatography from the 
aqueous methanol extracts of M. crenata and determined by HRESIMS, and 1H- and 13C 
NMR as loliolide and isololiolide. Loliolide and isololiolide inhibited the shoot and root 
growth of cress at concentrations greater than 10 and 30 μM, respectively, and for barnyard 
grass both allelochemicals started growth inhibition from 30 μM. There was an increase in 
inhibition as concentration of loliolide and isololiolide increased. The I50 values of cress 
and barnyard grass seedlings ranged from 32.1 to 128.5 μM for loliolide and 37.0 to 176.2 
μM for isololiolide. These results indicate that loliolide and isololiolide are main growth 
inhibitory substances which might bear responsibility for the M. crenata allelopathic 
potential.  
An aqueous methanol extract of R. maritimus  was subsequently purified by using 
chromatography and three growth inhibitory substances were determined by spectroscopic 
analysis as altechromone A, 5,7-dihydroxyphthalide and 2-methoxystypandrone. 
2-Methoxystypandrone, altechromone A and 5,7-dihydroxyphthalide exhibited significant 
inhibitory effects on the shoot and root growth of cress at concentration higher than 3, 100, 
3000 μM, respectively. The I50  values of 2-methoxystypandrone, altechromone A and 
5,7-dihydroxyphthalide for cress shoot and root growth were 5.8, 1406.7,1732.1 and 11.8, 
662.4, 2481.2 μM, respectively. The growth inhibitory results of the substances indicate 
that 2-methoxystypandrone, altechromone A and 5,7-dihydroxyphthalide might be potential 
allelochemicals in R. maritimus.  
In this research, four different weed species have shown strong allelopathic potential on the 
shoot and root growth of test plants, which indicates those weed may contain 
allelochemicals. Five potent allelochemicals were isolated and identified from M. crenata  
and R. maritimus  and those allelochemicals also displayed strong allelopathic effects on the 
growth of cress and barnyard grass seedlings. The study was the first attempt of finding out 
allelopathic potential in the weed species. Those findings might have greater value for weed 
allelopathy as well as crop-weed interference mechanism understanding and to use other 
strategies for weed management. 
 


English

Allelopathic Potential and Allelochemicals in Four Weed Species

Ehime University (愛媛大学)博士(学術)thesi

Vipa, Surojanametakul

（第３号様式）(Form No. 3) 
学  位  論  文  要  旨  
Dissertation Abstract 
 
 
氏名：  
Name 
 
          Vipa  Surojanametakul 
学位論文題目：  
Title of Dissertation 
 
Study on the remediation of Thai food safety with respect 
to food hypersensitivities 
(タイの食品への過敏症に対する安全性の改善策に関する研
究 )  
 
 
学位論文要旨：  
Dissertation Abstract   
The aims of these researches are; to investigate the undeclared food allergens in 
commercial Thai food product; to survey Thai commercial food products that have been 
reported to contain no wheat, rye, barley or gluten according to their labels; to 
investigate the understanding and implementation of food allergen management among 
Thai food manufacturers; to establish reliable enzyme-linked Immunosorbent assay for 
the determination of coconut milk proteins in processed foods. 
The presence of undeclared allergens – namely milk, egg, wheat and peanut – in 
142 commercial processed Thai food products were investigated by quantitative ELISA 
method. Fifty- five positive cases found to contain undeclared allergens > 10 ppm, 
wherein undeclared milk was the most frequently (38.2%) followed by egg and wheat 
with similar frequency (31% each). Undeclared peanut was rare. A significant number of 
cases containing undeclared food allergen greater than 1% were indicated to be wheat (5 
cases), egg (3 cases) and milk (3 cases). Confirmation test for the presence of egg 
(ovalbumin) and milk (casein) were also performed by Western blot test.  
Moreover, one hundred and twenty nine commercial Thai food products those 
have been reported to contain no wheat, rye, barley or gluten according to their labels, 
with respects to gluten free status, were surveyed if they were suitable for CD 
individuals by examining the absence of gluten or below 20 ppm. 
The gluten content in those products was analyzed using ELISA kit (LOQ 0.255 
ppm gluten). The results presented that most of the examined Thai food products 
(92.2%) contained less than 20 ppm gluten, which is the recognized safe gluten level for 
the Celiac disease (CD) individuals. However, 10 products found to contain >20 ppm 
gluten, among these 8 samples showed significant gluten presence exceeding 1,000 ppm. 
These 10 products showed clearly positive bands for wheat specific DNA by PCR 
analysis. The presence of undeclared gluten in examined products might be due to the 
use of undeclared wheat/gluten ingredients or cross-contact wheat/gluten. Our survey 
suggests that most of the examined Thai food products can be consumed by CD 
individuals, showed the potential of these Thai products as new diets to expand their 
（第３号様式）(Form No. 3) 
limited food choices from different food culture, and ultimately raise the quality of life 
of all CD individuals globally.  
Obviously, the two studies results revealed that appropriate allergen management 
strategies need to be implemented by Thai food manufacturer in order to ensure accurate 
labeling and to protect the allergic consumers. 
In the following study, we aim to learn about the understanding and 
implementation of food allergen management among Thai food manufacturers. Seventy 
two of HACCP-certified food companies were conducted by questionnaires and direct 
interviews with company’s QC managers, at period with no legal requirement on food 
allergen labelling. 55% (40/72) of respondents answered to implement a food allergen 
control program as part of existing product quality control and safety management. The 
awareness and understanding of food allergen management depended on the company’s 
dominance in the market place as well as experience in food quality control and safety 
management. High awareness of food allergy as well as the management and labeling of 
food allergens was found most in food manufacturers who sell their products oversea. 
The respondents expected that food allergen labeling legislation will soon be adopted in 
Thailand. The results of this survey indicated the substantial effectiveness of the present 
allergen control programs of Thai food manufacturers, and also their potential capability 
of food allergen management for domestic products at a level equal to that for export 
products. 
Additionally, a novel reliable Enzyme-Linked Immunosorbent Assay (ELISA) for 
detection and quantification of coconut milk proteins in processed foods designed and 
developed. The result demonstrated that developed sandwich ELISA was able to detect 
coconut milk proteins from various coconut milk products, and did not show any 
cross-reactivity with 41 of 42 kinds of popularly used food ingredients, thus reflecting 
great specificity for coconut milk proteins. Moreover, the established ELISA is highly 
sensitive and allowed the detection of 0.31µg/g of coconut milk protein in complex food 
matrices. This proposed assay could serve as a useful tool for detection of the presence 
of hidden coconut milk proteins in processed foods. 
 
 
 
 
 
 
 
 
 


Study on the remediation of Thai food safety with respect to food hypersensitivities

Chuan, He

（様式５）(Style5) 
学位論文全文に代わる要約 
Extended Summary in Lieu of Dissertation 
 
 
氏名： 
Name 
Chuan He 
 
学位論文題目： 
Title of Dissertation 
Studies on Ulvan-Degrading Enzymes from Alteromonas sp. 
（アルテロモナス属由来ウルバン分解酵素に関する研究） 
 
 
学位論文要約： 
Dissertation Summary 
 
General Introduction 
Energy shortages and environmental pollution have been affecting the sustainable development of the 
world. In order to solve the situation, marine macroalgal species have gained considerable global attention as 
source of renewable biomass. Among the three main divisions of macroalgae (Chlorophyta, Phaeophyta and 
Rhodophyta), green algae remain largely unexploited. In the past decade, marine eutrophication has promoted 
the proliferation of algal biomasses of Ulva sp. This is associated with an increase in human activity in coastal 
areas, which causes an increase in some nutrients in the aquatic environment, leading to growth of green algae, 
commonly known as algal blooms. One of the most spectacular happened along the shores of Qingdao just 
before the 2008 Olympic Games in Beijing, China. As a consequence of the strong environmental impact of 
these green seaweeds, the interest in exploiting such natural resource has increased significantly and in 
particular for its long chain polymer part, ulvan. 
The main constituents of ulvan are 3-sulfated rhamnose (R3S), D-glucuronic acid (GlcA), L-iduronic acid 
(IduA), and D-xylose (Xyl). The main structure subunit of ulvan are disaccharides comprising either GlcA or 
IduA linked to R3S, termed ulvanobiuronic acid A or B, respectively. In addition, disaccharide moieties made 
of R3S linked to Xyl occur in lower amounts (Fig. GI-1). As reviewed previously, the physicochemical and 
biological features of ulvan have many potential applications in food, pharmaceutical, agricultural, and chemical 
industries. 
Conversion from polymers into fermentable oligomers, requires chemical or biological break down. 
However, the process of chemical conversion could be very expensive and not worth-while to use for an 
economical large-scale production. The enzymatic hydrolysis has been suggested as a more environmentally 
and economically treatment due to the low energy requirement, less byproduct production. Thus, identification 
of ulvan-degrading microorganisms and of the corresponding enzymes is necessary to develop protocols for the 
bioconversion of ulvan. The objective of this study is to discover novel ulvan-degrading bacteria, which can 
produce ulvan-degrading enzymes and contribute to potential use of ulvan as oligomers. 
In this study, several enzymes involved in ulvan degradation were identified, heterologously expressed in 
（様式５）(Style5) 
E. coli, and characterized. A simple method for efficient renaturation of inclusion body proteins denatured by 
SDS were developed. 
 
Fig. GI-1. Structure of repeating disaccharide units in ulvan. Rhamnose 3-sulfate (Rha3S) is linked to (A) 
D-glucuronic acid (GlcA), (B) Liduronic acid (IdoA), (C) xylose (Xyl), or (D) xylose 2-sulfate (Xyl2S). (E) 
Nonreducing end after the cleavage of the glycosidic bond between Rha3S and GlcA or IduA via the 
β-elimination mechanism, 4-deoxy-L-threo-hex-4-enopyranosiduronic acid (Δ) linked to Rha3S. 
 
Long-type ulvan lyases in two Alteromonas sp. are major enzymes involved in degradation of ulvan 
extracted from Ulva ohnoi. 
1. INTRODUCTION 
Thus far, only a few ulvan-degrading enzymes have been isolated. The first ulvan lyase activity was found 
in a marine bacterium by Lahaye et al., who employed the newly discovered enzyme extract to degrade ulvan 
for structural analysis. Recently, a marine bacterium Persicivirga ulvanivorans sp. nov. (Re-classified as 
Nonlabens ulvanivorans) PLR was identified and its genome was sequenced. Two ulvan lyases were purified 
from N. ulvanivorans culture, sequenced, heterologously overexpressed in Escherichia coli, and the 
recombinant protein was biochemically characterized. These two ulvan lyases are 74% identical. This is the first 
ulvan lyase biochemically characterized. N. ulvanivorans ulvan lyase was reported to cleave ulvan at the 
β(1→4) glyosidic bond between R3S and GlcA or IduA via the β-elimination mechanism. The β-eliminative 
cleavage results in the formation of an unsaturated uronic acid (Δ, 4-deoxy-L-threohex-4-enopyranosiduronic 
acid) on the non-reducing end (Fig. GI-1E). Also, an N. ulvanivorans unsaturated β-glucuronyl hydrolase was 
found. This enzyme was shown to cleave specifically the unsaturated non-reducing end of the end products of 
（様式５）(Style5) 
the ulvan lyase. Most recently, two types, long and short, of ulvan lyase genes are identified from three 
Alteromonadales ulvan-degrading bacteria. Three strains contain very similar short ulvan lyases. The long-type 
ulvan lyase is composed of two modules, catalytic module, which is similar to short-type ulvan lyase, and the 
C-terminal module. Both short- and long-types of ulvan lyases exhibit enzyme activity. Ulvan lyases of N. 
ulvanivorans and three Alteromonadales bacteria show no sequence similarity. 
In this study, two ulvan lyases were identified from two ulvan-utilizing Alteromonas sp., heterologously 
expressed in E. coli, and characterized.  
 
2. MATERIALS AND METHODS 
2.1 Screening and isolation of ulvan-utilizing bacteria 
Mollusks and shrimps were collected at a pond near Usacho Inoshiri, Tosa, Kochi Prefecture, Japan, and 
fed in the lab with Ulva ohnoi using ASW. Their feces were collected and used as sources of ulvan-utilizing 
bacteria. Feces were inoculated into half strength marine broth (1/2 MB, 18.7 g Difco Marine broth 2216 and 
9.8 g NaCl in 1000 ml DW) containing 0.2 % ulvan and incubated under agitation at 25 °C for overnight. Cell 
suspension was spread on ulvan agar media (0.2 % ulvan in three-quarter strength artificial seawater with 1.5% 
agar) and incubated at 25 °C for several days. Colonies were replicated onto both 1/2 MB agar and ulvan agar 
media. After cells grew, the ulvan agar media were flooded with 5% CPC. While CPC reacted with acidic 
polysaccharides, such as ulvan, to form insoluble precipitates, clear haloes were visible when bacterial cells 
degraded ulvan to oligosaccharides. The diameter of the halo is a measure of the amount of degradation activity 
produced. The bacteria that degraded the ulvan agar media were considered as ulvanolytic. 
2.2 Extraction of secreted ulvan-degrading enzymes 
Ulvan-utilizing bacterial cells grown in 40 ml of 1/2 MB were transferred to 4 l of fresh 1/2 MB containing 
0.05% ulvan and cultivated at 25 °C for 3 days in a mini jar fermentor (TSO-M, Takasaki Kagaku). Bacterial 
culture supernatant was prepared by centrifugation at 12,000 x g for 30 min, and ammonium sulfate was added 
to 70% saturation to extract secreted proteins. The precipitate was suspended in the small volume of 20 mM 
Tris-HCl (pH 8.0) and dialyzed against the same buffer. In a small volume of culture supernatant, an 
ultrafiltration concentrator (Vivaspin 500, Sartorius) was used. 
2.3 Purification of crude ulvan-degrading enzymes 
Proteins precipitated with ammonium sulfate were loaded onto 10 ml of Q Sepharose Fast Flow (GE 
Healthcare) equilibrated with 20 mM Tris-HCl buffer (pH 8.0), and bound proteins were eluted with a 100-ml 
linear gradient of NaCl (0-0.5 M) in the same buffer. Proteins in each fraction were analyzed on SDS-PAGE, 
and active fractions containing ulvan-degrading enzymes were identified using ulvan-gel overlay assay 
indicated in section 2.6.  
Active fractions were pooled, and ammonium sulfate was added at a final concentration of 2 M. This 
solution was loaded onto 10 ml of Phenyl Sepharose 6 Fast Flow (GE Healthcare) equilibrated with the buffer 
(20 mM Tris-HCl (pH 8.0) and 2 M ammonium sulfate). Bound proteins were eluted with a 100-ml linear 
（様式５）(Style5) 
gradient of ammonium sulfate (2-0 M) in 20 mM Tris-HCl (pH 8.0). Active fractions were assayed as above 
and concentrated by using Vivaspin 500. 
2.4 Activity staining overlaid on ulvan-containing agarose gel 
We adopted the protocol for alginate lyase with the following modifications. Proteins were separated on 
SDS-PAGE, and enzymes were renatured by incubating the gel three times in 100-ml of 20 mM Tris-HCl (pH 
8.0) for 10 min. The polyacrylamide gel was overlaid onto an agarose gel (0.2 % ulvan in three-quarter strength 
artificial seawater with 1.0% agar) and incubated overnight at 30 °C. The agarose gel was immersed into 5% 
CPC, and ulvan-degrading activities were visualized as clear bands. 
2.5 Identification of ulvan lyase genes 
Proteins exhibiting ulvan-degrading activities were analyzed on SDS-PAGE and transferred to a PVDF 
membrane using the blotting buffer (10 mM CAPS (pH 11.0) and 10% methanol) at 4 °C. Proteins were stained 
with CBB R-250 and excised from the membrane. N-terminal amino acid sequences of the excised proteins 
were determined using protein sequencer PPSQ-31A (Shimadzu). In order to identify genes encoding the 
proteins, the determined amino acid sequences were searched against the protein database deduced from draft 
genome sequences of ulvan-utilizing strains using BLASTP program. 
2.6 Cloning of ulvan lyase genes 
Full length of long-type ulvan lyase gene (ullA) of KUL17 strain was PCR amplified with primers of 
17ullAA1 and 17ullAB1 from chromosomal DNA of KUL17 using PrimeSTAR HS DNA polymerase (Takara 
Bio). C-terminal truncated versions of ullAKUL17 were PCR amplified with primer pairs of 17ullAA2 and 
17ullAB2 and 17ullAA2 and 17ullAB3, respectively. Full length of long-type ulvan lyase gene (ullA) of 
KUL42 strain was PCR amplified with primers of 17ullAA1 and 42ullAB1 from chromosomal DNA of 
KUL42. C-terminal truncated versions of ullAKUL42 were PCR amplified with primer pairs of 17ullAA2 and 
42ullAB2 and 17ullAA2 and 42ullAB3, respectively. All the PCR products were cloned into pET21a using 
In-Fusion HD Cloning Kit (Takara Bio). The nucleotide sequences of ullAKUL17 and ullAKUL42 have been 
deposited in DDBJ under accession numbers LC278382 and LC278383, respectively. 
2.7 Purification of ulvan lyases 
Escherichia coli BL21(DE3) was transformed with plasmids and ulvan lyases were over expressed in the 
transformant cells by adding 1 mM IPTG as previously described. After IPTG addition, cells were incubated at 
20 °C for 4 hr. Cells were harvested and disrupted by sonication. After removing cell debris, the supernatant 
was used for Ni2+ affinity column (Ni-NTA Agarose, Qiagen) purification. 
 
3. CONCLUSION 
Two ulvan-utilizing Alteromonas sp. with high ulvan degrading activity, KUL17 and KUL42, 
were isolated from feces of small marine animals. We identified ulvan lyases with ca. 55 kDa in size 
from the secreted proteins of two strains and cloned the genes for them. The predicted molecular 
weight of the long-type ulvan lyases is 110 kDa. The catalytic domain is located on the N-terminal half 
（様式５）(Style5) 
and the C-terminus was turned out to be deleted. Although both strains contain two ulvan lyases, long 
and short, long ulvan lyase was demonstrated to be the major lyase for ulvan degradation. 
 
Efficient renaturation of inclusion body proteins denatured by SDS 
1. INTRODUCTION 
When target proteins are over expressed ectopically, such as in Escherichia coli, proteins 
sometime form inclusion bodies. Inclusion body proteins are basically biological inactive. For getting 
active proteins, we need either to find culture conditions without inclusion bodies, such as at lower 
temperature, or to solubilize proteins in inclusion bodies by some methods.  
The purification of the expressed protein from inclusion bodies requires three steps, extraction of 
inclusion bodies from cells, solubilization, and refolding. Inclusion bodies are easily isolated from cells 
by low-speed centrifugation because inclusion bodies have higher density. Inclusion bodies are 
solubilized by high concentration of chaotropic reagents, such as urea and guanidine hydrochloride. 
Solubilized proteins are refolded by gradual removal of chaotropic reagents. Refolding is 
time-consuming and not all the solubilized proteins are renatured due to protein aggregation in the 
process of refolding. 
Mild detergents, such as sarkosyl, are also used for solubilization. Sarkosyl works better than the 
chaotropic reagents in terms of aggregation in the refolding process. In this study, we have used a 
strong ionic detergent SDS for solubilization of inclusion body proteins and refolded proteins by 
removing SDS at lower temperature with KCl. Suzuki and Terada reports the removal of SDS from 
protein solution by the addition of potassium salts. Ulvan lyase from Glaciecola sp. KUL10 formed 
inclusion body when it was over expressed in E. coli at any condition. SDS-solubilized ulvan lyase was 
successfully renatured as an active form by removing SDS. 
 
2. MATERIALS AND METHODS 
2.1 Cloning of ulvan lyase gene and expression in E. coli 
Full length of ulvan lyase gene ullA without signal sequence was PCR amplified from 
chromosomal DNA of ulvan-degrading Glaciecola sp. KUL10 (unpublished) with primers, 10ulhAA5 
(5′-GGAGATATACATATGGATGTCGTGCTTGAAAAACA-3′) and 10ulhAB3 
(5′-GTGGTGGTGGTGGTGTTATGATGCACAACGCGGAC-3′), and cloned into pET21a. The 
C-terminal truncated version of ullA was amplified with 10ulhA5 and 10ulhB5 
(5′-GTGGTGGTGGTGGTGAGCTTCACTTTCCAAATCTA-3′) and cloned in the same vector, to make 
pullA5. This truncated version contains His-tag on the C-terminus. E. coli BL21(DE3) was 
transformed with pullA5 and ulvan lyase was over expressed in the transformant cells by adding 
IPTG as previously described. The nucleotide sequence of ullA has been deposited in DDBJ under 
accession number LC278381. 
（様式５）(Style5) 
2.2 Refolding of ulvan lyase in inclusion bodies 
Cells containing over-expressed ulvan lyase were disrupted by sonication. Inclusion bodies were 
recovered by centrifugation at 10,400 x g for 30 min at 4 °C. The pellet was washed three times with 
20 mM Tris-HCl (pH 8.0), suspended in the denaturation buffer (50 mM Tris-HCl (pH 8.0), 300 mM 
NaCl, 2 % SDS), and sonicated until it became clear. Excess SDS was removed at 4 °C at least for 30 
min to overnight. After centrifugation at 10,400 x g for 10 min, KCl was added to the supernatant at 
the final concentration of 400 mM. SDS-KCl insoluble crystal was formed overnight at 4 °C. The 
supernatant after centrifugation as above was used for Ni2+ affinity column (Ni-NTA Agarose, Qiagen) 
purification. 
2.3 Enzyme assay for ulvan lyase 
Ulvan was extracted from Ulva ohnoi by hot water. 0.1 % (w/v) ulvan solution was incubated with 
0.2 µg purified enzyme in the buffer (20 mM Tris-HCl (pH 8.0), 500 mM NaCl). The formation of 
double bonds was monitored by the increase in absorbance at 235 nm. The formation of reducing ends 
was measured by DNS method. Enzyme reaction was carried out in 500 µl of solution (0.2 % (w/v) 
ulvan, 20 mM Tris-HCl (pH 8.0), 500 mM NaCl, and purified enzyme) for 1 hr at 25 °C. After adding 
500 µl of DNS reagent (1 % dinitrosalicylic acid, 30 % potassium sodium tartrate, 0.4 M NaOH), the 
mixture was boiled for 10 min and cooled, and the amount of reducing ends was monitored by 
measuring absorbance at 540 nm. L-Rhamnose was used to make a standard curve. 
 
3. CONCLUSION 
Inclusion bodies are often formed when the foreign protein is over expressed in Escherichia coli. 
Since proteins in inclusion bodies are inactive, denaturing and refolding of inclusion body proteins are 
necessary to obtain the active form. Instead of the conventional denaturants, urea and guanidine 
hydrochloride, a strong anionic detergent SDS was used to solubilize C-terminal His-tag form of ulvan 
lyase in the inclusion bodies. Solution containing SDS-solubilized enzyme were kept on ice to 
precipitate SDS, followed by SDS-KCl insoluble crystal formation to remove SDS completely. After 
removing the precipitate by centrifugation, the supernatant was applied to Ni-NTA column to purify 
His-tagged ulvan lyase. The purified protein showed a dimeric form and ulvan lyase activity, 
demonstrating that SDS-denatured protein was renatured and recovered enzyme activity. This simple 
method could be useful for refolding other inclusion body proteins. 
 
 
 
 


Studies on Ulvan-Degrading Enzymes from Alteromonas sp.

ワーキングペーパーjournal articl

山口, 由等

Japanese

地域レジリエンスと四国の鉄道文化・産業遺産

Ehime University (愛媛大学)博士(医学)thesi

神崎, 博充

KANZAKI, Hiromitsu

（第 12号様式） 
 
 
学 位 論 文 の 要 約 
（ 研 究 成 果 の ま と め ） 
 
 
 
            氏   名  
神崎 博充 
 
            学位論文名  
根治的放射線治療を施行された病期 III 期非小細胞肺癌における組織学的な亜型による早期の腫
瘍縮小の治療成績への影響の違い 
 
    学位論文の要約 
病期 III 期非小細胞肺癌において、病期学的亜型による治療経過中の腫瘍縮小率と治療効果との
関連の違いについて検討した[Kanzaki, Kataoka and Nishikawa, 2016: 主論文]。また、前立腺
癌に対する密封小線源治療において、PSA bounce と PSA 再発との間の PSA 変化の違いについて
検討した[Kanzaki, Kataoka and Nishikawa, 2015: 参考論文]。 
 
主論文：Kanzaki H, Kataoka M, Nishikawa A: Impact of early tumor reduction on outcome 
differs by histological subtype in stage III non-small-cell lung cancer treated with d
efinitive radiotherapy. International journal of clinical oncology 21(5): 853-861, 201
6 DOI: 10.1007/s10147-016-0982-0 
参考論文：Kanzaki H, Kataoka M, Nishikawa A: Kinetics differences between PSA bounce 
and biochemical failure in patients treated with 125I prostate brachytherapy. Japanes
e journal of clinical oncology 45(7) 688-694, 2015 DOI: 10.1093/jjco/hyv050 


Impact of early tumor reduction on outcome differs by histological subtype in stage III non?small?cell lung cancer treated with definitive radiotherapy

Ehime University (愛媛大学)博士(医学)doctoral thesi

川崎, 俊

KAWASAKI, Shun

Effects of hypnotic bromovalerylurea on microglial BV2 cells

Muhammad, Novrizal Abdi Sahid

（第７号様式） 
 
学位論文審査結果の要旨 
 
   氏     名   
 
 
 
 
 
   審 査 委 員 
 
 
 
 
 
 
 
 
 
                                      
 
                                      
 
                                      
 
                                      
 
                                      
 
 
      論 文 名   
スタチンによるメバロン酸経路阻害はマスト細胞の脱顆粒反応を抑制する 
 
審査結果の要旨（2,000 字以内） 
 
学位論文の要旨 
［背景と目的］マスト細胞からのヒスタミンの遊離はⅠ型アレルギー反応を惹起するが、こ
の遊離の過程を薬剤によって制御できれば多くの患者を治療できると期待される。HMG-CoA
還元酵素阻害剤として知られるスタチン製剤は、高コレステロール血症の治療に用いられる
一方、免疫系に作用して炎症症状の改善に関わることが明らかになっている。そこで本研究
では、培養細胞を用いて、スタチン投与によってマスト細胞からのヒスタミンの開口分泌が
阻害されるかどうかを検討し、その阻害メカニズムを明らかにしようとした。 
［材料と方法］マスト細胞の腫瘍細胞系であるラット好塩基球性白血病細胞（RBL-2H3）を
用いて、抗原（DNP-BSA）、カルシウム・イオノフォア（ionomycin）、小胞体の Ca-ATPase
阻害薬（thapsigargin）の３種の刺激によってヒスタミン遊離を誘導し、その反応がスタチ
ンの一種である simvastatin の前処理によって抑制されるかどうかを検証した。さらにその
メカニズムを明らかにするため、geranylgeraniol 処理による抑制の解除を検討した。開口
分泌に関わる分子についてはその mRNA やタンパク質の発現を検討し、低分子量Ｇタンパク
の一種 Rab27a と Ca 結合タンパクの一種 Doc2a が重要な分子であると予想されたため、両者
の相互作用を市販のキットを用いた Proximity ligation assay によって解析した。 
［結果と考察］マスト細胞の培養系を上記の３種いずれの方法で刺激しても、simvastatin
はヒスタミン遊離を抑制した。このことから、simvastatin が阻害している過程は刺激から
遊離に至る経路の最終段階であると予測された。検討した４種のスタチンの中では、simva-
statin が最も有効であると考えられた。また、simvastatin による抑制は geranylgeraniol
処理によって回復したことから、タンパク質のゲラニルゲラニル化の阻害がメカニズムとし
て重要であることが示された。さらに、開口分泌を制御しているタンパク質からゲラニルゲ
ラニル化のターゲットを同定するため、その候補について mRNA の発現を PCR 法にて、タン
パク質の細胞内局在を蛍光抗体法にて、それぞれ確認した。最後に、Doc2a と Rab27a との
タンパク質同士の相互作用を、Proximity ligation assay 法によって示した。 
 simvastatin は、Rab27a 等の開口分泌に関わる分子のゲラニルゲラニル化を抑制すること
によって、マスト細胞からのヒスタミン遊離を阻害すると考えられた。本研究により、スタ
チンによるアレルギー反応抑制作用とその分子メカニズムの一端が明らかにされた。 
 
審査結果の要旨 
 本論文の公開審査会は、平成２９年２月２日に行われた。 
 申請者は、今回の研究の目的と方法、結果とその解釈について英語で説明した。その後、
審査委員から英語と日本語で質問を行い、申請者はこれらの質問に対して英語で応答した。
主な質問内容は以下の通りであった（［ ］内は申請者の応答）。 
・スタチンのアレルギー反応抑制について、ヒトや他のモデルでの検討事例はあるか。
［癌患者の例があるが、アレルギーについては報告されていないと思う。］ 
・使用した simvastatin はプロドラッグか。［分子構造の変化した活性型を用いた。］ 
・他の阻害剤の効果は検討したか、スタチンの脱顆粒抑制以外の効果は考えられるか。
［行った実験の範囲では、細胞傷害性は可能性としてありえる。］ 
・IgE 抗体の抗原性の記載は正確か。［以前に所属研究室が開発した抗体を用いた。］ 
・実験によってコントロール群でもヒスタミン遊離の量が異なるのはなぜか。［細胞株の
継代数の違いや培養条件等によって、実験条件が一定しなかった。］ 
・スタチンの種類による効果の違いは、由来や水溶性の違いによるものか。［分子構造に
よると考えられるが、単純な分類による違いではなかった。］ 
・ヒスタミンの遊離量と含有量との関係が矛盾していないか。［矛盾ではないと思うが、
スタチンによる細胞傷害の影響が見られる。］ 
・ヒスタミンの delayed release の可能性を検討したか。［今回は検討していない。］ 
・ヒスタミン遊離抑制の解除に farnesol が無効な理由は何か。［代謝経路が farnesol で
分岐しているので、予期したのとは別の方向に代謝されてしまった可能性がある。］ 
・simvastatin のターゲットは Rab27a だけか。［開口分泌はタンパク質複合体で制御され
ているので、cdc42 等の他のタンパク質も候補である。］ 
 
学位論文審査の結果 
 以上から、審査委員会は、申請者が研究テーマとその関連領域に対して学位授与に値する
十分な見識と能力を有することを全員一致で確認し、本論文が博士（医学）の学位授与に値
すると判定した。 


Inhibition of the mevalonate pathway by simvastatin interferes with mast cell degranulation by disrupting the interaction between Rab27a and double C2 alpha proteins

Zhang, Xin

 
 
（第５号様式）  
                        学位論文審査の結果の要旨 
 
    氏    名 
   Zhang Xin 
 
審 査 委 員 
       
 
     
 論  文  名 
   Study on the Palatability of Chinese Japonica-type Rice Varieties   
      (中国産ジャポニカ型水稲品種の食味に関する研究) 
 
    審査結果の要旨 
中国では近年，経済発展に伴う食生活の多様化によって，消費者の間では品質や食味に優れた
ジャポニカ米に対する要望が強くなってきている．しかし，現在の中国では米の食味についての
研究は少なく，食味を目的とした品種育成もほとんど行われていない．こうした背景の中で，本
研究は中国産ジャポニカ型水稲品種の食味特性を明らかにし，中国における科学的な食味評価法
と効率的な良食味品種育成法を開発することを目的に行われた． 
 中国人消費者の食味に対する嗜好性を把握するために，中国および日本産の10品種を用いて中
国人パネルと日本人パネルによる日本式の食味官能試験（外観，味，粘り，硬さ，総合評価）を
実施した．その結果，中国人パネルの総合評価と日本人パネルの総合評価との間には有意な正の
相関関係が存在し，全体的にみると，中国人パネルと日本人パネルの米に対する嗜好性は大きく
変わらないと推測された．ただし，日本人パネルの食味評価は極めて低いにも関わらず，中国人
パネルの評価はそれほど悪くないという品種が幾つかあった．また，総合評価に対する各食味評
価項目の貢献度を比較したところ，日本人パネルでは中日両国品種とも粘りと外観を重視してい
たが，中国人パネルは中国産品種では硬さと粘り，日本産品種では味と外観を重視していた．ま
た，中国人パネルは硬い米，日本人パネルは軟らかい米を好む傾向がみられた．したがって，日
本式の官能試験法を中国に適用する場合，これらについて注意を払う必要があると考えられた． 
 中国各地から収集した260品種について，理化学的特性（アミロース含有率，タンパク質含有率，
最高粘度，ブレークダウン，硬度/粘度比）の遺伝変異とその遺伝相関を解析した．260品種の理化
学的特性には，日本の良食味米基準を超える高いレベルから極端に低いレベルまでの幅広い遺伝変
異が存在した．タンパク質含有率とアミロース含有率との間に負，タンパク質含有率とブレークダ
ウンとの間に正の，いずれも食味にとって好ましくない遺伝相関が認められた．これらより，中国
産品種は理化学的特性のバランスが悪く，特にタンパク質含有率に問題があることが判明した． 
 
 
 中国および日本産各10品種の理化学的特性を比較するとともに中国産品種の理化学的特性におけ
る問題点について検討した．両国品種のアミロース含有率に有意差はなかったが，中国産品種は日
本産品種よりも最高粘度とブレークダウンが有意に低く，タンパク質含有率と硬度/粘度比が有意に
高かった．アミロース含有率とタンパク質含有率との間には，日本品種では食味にとって好ましい
正の相関傾向が認められたが，中国品種では逆に有意な負の相関関係が存在した．したがって，中
国品種ではタンパク質含有率の改善，特にタンパク質含有率とアミロース含有率との間の負の遺伝
相関を破ることが食味育種にとって最も重要な課題であると判断された． 
 中国産米のタンパク質含有率が高い理由の一つは，栽培に大量の窒素肥料が施用されていること
である．そこで，天津地域の基幹品種である津川1号と塩豊47を供試し，窒素施用量と理化学的特
性および収量との関係を解析した．窒素施用量の増加に伴ってアミロース含有率，最高粘度，ブレ
ークダウンは低下し，タンパク質含有率は高くなった．このため，食味計で測定した食味値は窒素
施用量が多いほど低下する傾向がみられた．両品種とも窒素施用量が多いほど穂数が増加し，収量
は向上した．食味と収量を総合してみた最適窒素施用量は15-20gNm-2程度と推定された． 
中国の北方地域で生産された65品種について理化学的特性と炊飯特性（吸水率，膨張率，延伸
率）の品種間相関について解析した．その結果，理化学的特性と炊飯特性との間に有意な相関関係
はみられず，理化学的特性によって炊飯米の特性を推定することは難しいと考えられた． 
日本では，理化学的特性を選抜指標にした効率的な食味育種法が確立されているが，中国では理
化学的特性と実際の食味との関係はほとんど検討されていない．そこで，中国産の28品種を用いて，
官能試験の各評価項目と理化学的特性との関係を解析した．総合評価，外観，香り，味および粘り
とタンパク質含有率および硬度/粘度比との間に負，最高粘度およびブレークダウンとの間に正の有
意な相関関係が認められ，総合評価の品種間差の約47％がタンパク質含有率，最高粘度および硬度/
粘度比によって説明できると推測された．これらより，中国においても日本式の選抜法すなわち多
くの系統を扱う初期世代では理化学的特性に基づいて大まかな選抜を行い，系統数が絞られてきた
中後期世代から官能試験を行うことで良食味系統を効率的に選抜できる可能性が示された．   
 天津農学院の中日水稲品質・食味共同研究センターで育成された高度良食味品種津川1号の栽培
適地の拡大すなわち他の省への導入と普及が可能かどうかを確認するために，天津市と江蘇省宝応
県で栽培して収量，収量構成要素，理化学的特性を調査した．栽培地を高緯度地域（天津市）から
低緯度地域（江蘇省）へ移動することで栄養生長期間が短縮し，移植期から穂揃期までの日数が短
くなった．宝応県での収量は天津市に比べて12％減少した．一方，理化学的食味特性は，宝応県で
栽培した方がアミロース含有率とタンパク質含有率が低く，最高粘度が高くなり，食味は向上する
傾向がみられた．これらより，津川1号は収量が低いために江蘇省に直接導入することは難しいが，
良食味品種育成の交配母材として活用できると考えられた． 
 
 本論文の公開審査会は平成29年2月4日に愛媛大学農学部で開催され，論文発表と質疑応答が行な
われた．引き続いて学位論文審査会を開催して審議した結果，審査委員全員一致して本論文が博士
（農学）の学位を授与するに値すると判定した． 
 
 
 


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