Kochi University Repository

Tse-Min, Lee

Tsure-Meng, Wu

Ming-Shiuan, Sung

Yuan-Ting, Hsu

Hsueh-Ling, Chang

Cheng-Yang, Kang

Yi-Ting, Hsu

Kuan-Lin, Ho

115
Kuroshio Science 6-1, 115-119, 2012
Abstract
1. Introduction
Ulva as a Model for the Study of Environmental stress in Intertidal 
Macroalgae
Tse-Min Lee*, Tsure-Meng Wu, Ming-Shiuan Sung, Yuan-Ting Hsu, Hsueh-Ling 
Chang, Cheng-Yang Kang. Yi-Ting Hsu, Kuan-Lin Ho
Institute of Marine Biology, National Sun Yat-sen University, Kaohsiung 804, Taiwan
Doctor Degree Program in Marine Biotechnology, National Sun Yat-sen University, 
Kaohsiung 804, Taiwan
The Kuroshio Research Group of the Asia-Pacific Ocean Research Center, National
 Sun Yat-sen University, Kaohsiung 804, Taiwan
*Corresponding author: e-mail  tmlee@mail.nsysu.edu.tw
sensing the outer salinity changes have now been developed.
Key words: acclimation, hypersalinity, oxidative stress, Ulva fasciata
The macroscopic chlorophyte, Ulva fasciata Delile, is abundant in shallow waters 
and intertidal zones along the Taiwan coast, where light intensity can increase to 
- -12around 1,800    mol m  s  at noon, particularly in the summer season and salinity 
can increase to around 60-80%  in the intertidal pools. A series of experiments were 
therefore initiated in our laboratory to study how intertidal U. fasciata acclimates to 
high salt stress at the molecular level. We have identified the regulation of ion 
balance and proline synthesis by Ca and calmodulin via a modification of proline 
dehydrogenase activity in U. fasciata, and the up-regulation of genes encoding 
protein degradation enzymes and heat shock protein 90 for intertidal green 
macroalga Ulva fasciata against hypersalinity-induced protein oxidation. To cope 
with hypersalinity, the expression of genes of antioxidant enzymes is up-regulated 
in U. fasciata against oxidative stress. The signals and the following network in 
      Intertidal macroalgae frequently suffered from 
daily fluctuations in light intensity, desiccation, 
salinity, temperature, and UV exposure (Lobban 
and Harrison 1997; Kakinuma et al. 2006). The 
mechanism dealing with the acclimation and 
adaptation to stressful environments is critical for 
the survival of intertidal macroalgae. Because 
macroalgae do not have the advantage of motility 
like animals, they have developed mechanisms to 
cope with acute environmental changes, for 
example, salinity elevation in the intertidal pool. 
Our preliminary study noted that a marine 
macroscopic chlorophyte, Ulva fasciata Delile, 
was abundant in shallow waters and intertidal 
zones along the Taiwan coast, where salinity can 
increase to around 60-80%  in the intertidal pools 
initiated in our laboratory to study how intertidal 
U. fasciata acclimates to high light and high salt 
stress at the molecular level. Our results in the past 
decade were summaried as follows.
 
Fig. 1. Ulva fasciata Delile is abundant along the 
            costal shore in Taiwan.
(Fig. 1). A  series  of  experiments  were  therefore 
116
Ulva as a model for the study of environmental stress
Liu. 1999. Regulation of NaCl-induced proline 
-++Fig. 2. Changes in Na  (A), K  (B), and Cl  (C) 
            in Ulva fasciata in response to elevated 
            ASW salinity by increasing NaCl concent-
            ration in ASW (open bar) or concentrating 
            ASW (solid bar). Vertical bar indicates the 
            standard error (n = 3) (Lee and Liu, 1999)
     
     It is considered that the control of constant cell 
turgor by regulating osmotic potentials through 
the adjustment of ion and organic osmolyte con-
tents, is a typical tolerance mechanism observed 
in most marine algae (Kirst, 1990). When algae 
are exposed to salinity changes, the movement of 
water occurs first, followed by ion fluxes for the 
maintenance of constant cell turgor by regulating 
+osmotic potential; monovalent ions including Na , 
-+K  and Cl  are the amin ionic osmolytes contri-
buting to the osmotic adjustment (Kirst, 1990). 
After that, the number of organic osmolytes, such 
as proline, accumulated in algae in response to 
salinity, changes. We first examined how U. 
fasciata counteracted the hypersalinity stress 
through the maintenance of water. These results 
were published between 10-12 years ago (Lee and, 
 
accumulation by calmodulin via a modification of 
proline dehydrogenase activity in Ulva fasciata 
(Chlorophyta). Australian Journal of Plant 
Physiology 26: 595-600; Lee TM, Liu. 1999). 
Correlation of decreased calcium contents with 
proline accumulation in the marine green 
macroalga Ulva fasciata exposed to elevated NaCl 
 
Journal of Experimental Botany.50:1855-1859. 
+ +     Lee and Liu (1999) have found that Na , K , 
-
and Cl  concentrations can be increased linearly 
as salinity increases in U. fasciata (Fig. 2). 
-+ +
It is evident that Na , K , and Cl  are used in U. 
fasciata for the control of osmotic potential upon 
exposure to hypersalinity. It was also found that 
the increase of salinity by the addition of NaCl led 
to a decrease in intrace-llular total and soluble 
2+Ca  concentrations but an increase in medium 
2+ +2Ca  concentration, reflecting a efflux of Ca . 
Therefore, hypersaline treatment by concentrated 
+2seawater did not influence Ca  concentrations. 
Our study showed that calcium effluxes in U. 
fasciata upon exposure to high salinity conditions.
    The elevation of salinity by the addition of 
NaCl induced proline accumulation in Ulva 
fasciata via increased synthesis and decreased 
content  in  seawater;  which  is  published  in 
2. #Regulation of ion balance and proline synth-
   esis in Ulva fasciata in response to salinity 
   stress
Fig. 3. Changes in proline in Ulva fasciata in 
            response to elevated ASW salinity by 
            increasing NaCl concentration in ASW. 
            Solid circle: 55 %  , Open circle: 30 %  . 
            Vertical bar indicates the standard error (n 
          = 3) (Lee and Liu, 1999).
degradation  (Fig. 3). Evidence  suggests  that 
117
pathway in the modulation of proline synthesis and 
degradation, and in turn, proline accumulates.
    hypersalinity
calcium is the factor in the regulation for proline 
synthesis for U. fasciata against hypersalinity 
stress. The activity and Vmax value of P5C 
reductase were increased by hypersalinity, while 
proline  dehydrogenase (PDH)  and P5C  dehydro-
genase (P5CDH) activities were significantly 
decreased, accompanied by an increase in Km 
2+value. Both a decrease in Ca  availability and a
block of calmodulin action cause a decrease in 
PDH activity and and an increase in proline con-
centration. These suggest that when exposed to 
2+hypersalinity (NaCl), a loss of Ca  from Ulva 
fasciata cells alters the calmodulin-mediated 
 
3. Molecular acclimation of Ulva fasciata to 
   
   A forward cDNA library was constructed via the 
suppressive subtractive hybridization between 
30%  and 90%  (24 h) and by the time-course 
dynamics of several abundantly expressed genes.
    Among the genes with known sequences, the 
expressed sequence tags (ESTs) are abundant in 
the function of protein synthesis (ribosomal 
protein) and destination (Fig. 4). The cDNAs of 
ATP-dependent Clp protease, (UfClpC), 20S 
proteasome B subunit type 1 domain (UfPbf1), 
ubiquitin-conjugating enzyme E2 I (UfUbc9), and 
heat shock protein 90A (UfHsp90A) were cloned. 
UfClpC transcript increased 3 h after 90%   treat-
ment, followed by a decrease, while UfPbf1 and 
UfUbc9 transcripts increased after 12 h and 
decreased at 48 h. The transcripts of UfHsp90A 
increased 1 h after 90%  treatment, followed by a 
drop and to the control level at 48 h. Protease 
activity increased 3 h after 90%  treatment and 
decreased to the control level at 48 h (Fig. 5). 
H2O2 contents increased 1 h after 90%   treatment 
and then remained unchanged but protein carbonyl 
group contents increased after 48 h. The treatments 
of reactive oxygen species scavengers partially 
alleviated 90%   damage (partial growth rescue) 
and suppressed the increases in H2O2 content, 
protein carbonyl group content, protease activity, 
and UfClpC, UfPbf1, UfUbc9, and UfHsp90A 
transcripts by 90% . The induction of specific 
chaperones and proteases at the molecular level 
for protein quality control can be considered as 
one of molecular mechanisms of hypersalinity 
acclimation in U. fasciata. 
Fig. 4. The functional grouping of forward cDNA 
            library via the suppressive subtractive 
            hybridization between 30%   and 90% 
           (Sung et al. 2010). (24 h) 
Implications of the up-regulation of genes encoding 
protein degradation enzymes and heat shock protein 
Marine Biotechnology 13: 684-694.). SSH has been 
survival of U. fasciata in the removal of oxidatively 
90%    grown thalli to scavenge overproduced ROS, 
genes were examined.
     In attempts to identify the whole picture of U. 
fasciata to hypersalinity stress, the transcripteomics 
analysis has been carried out using suppression 
subtractive hybridization (SSH) (Sung et al., 2010. 
90 for intertidal green macroalga Ulva fasciata 
against hypersalinity-induced protein oxidation. 
used to extract differentially expressed genes in 
algae in response to hypersalinity. In this study, 
mRNAs from 30%  (control) and 90%  (hypers-
alinity) treatments (24 h) were extracted for the 
construction of the SSH library to elucidate the 
whole picture of metabolism responses to 
hypersalinity in U. fasciata at the molecular level. 
We  found that the SSH cDNAs for protein 
synthesis and destination were abundant. From 
these, several genes were selected for cloning of 
the full-length cDNAs and the examination of 
time course changes in mRNA expression levels 
in relation to protein degradation. In attempts to 
explore the role of these genes in the hypersalinity 
 
modified proteins, ROS scavengers were treated to 
and the recovery growth ability as the growth rate 
after recovery to 30%  condition, H2O2 and protein 
carbonyl group contents, and mRNA levels of these 
Tse-Min Lee, Tsure-Meng Wu, Ming-Shiuan Sung et al.
118
Ulva as a model for the study of environmental stress
Fig. 5. Time course changes in the transcripts of 
UfClpC (a), UfPbf1 (b), UfUbc9 (c), and 
UfHsp90A (d) (means ± SD, n=3) of U. fasciata 
exposed to 30%  or 90%  . The relative transcript 
abundances were assayed by the detection of SYBR 
Green fluorescence in quantitative real-time PCR 
and presented as the fold change in mRNA abund-
ance, normalized to an endogenous reference gene 
(α-tubulin; GenBank no. EU701065), relative to 
the RNA sample of 30% -grown U. fasciata at 0 h. 
Asterisk indicates significant difference by t-test 
(Sung et al. 2010)
regulated against salinity stress.
4. Induction of antioxidant defense system in U. 
    faciata against oxidative stress in hypersalinity 
    stress.
    Because one of EST genes, ascorbate peroxidase,  
as the antioxidant enzyme, has been found in U. 
fasciata after 24 h of exposure to hypersalinity, 
we proposed that antioxidant enzymes may be 
involved in U. fasciata in the defense of oxidative 
stress. In addition, our previous study, (Lu IF, 
Sung MS, Lee TM. (2006) Salinity stress and 
    We have identified that hypersalinity (60%  , 
90%  ) as well as hyposalinity (15%  ) caused lipid 
peroxidation and H2O2 accumulation, and that a 
H2O2 scavenger, dimethylthiourea, effectively 
inhibited H2O2 increase and oxidative stress. 
Antioxidants and the enzyme activity and mRNA 
expression levels of several antioxidative enzymes, 
including superoxide dismutase (SOD) (Fig. 6), 
ascorbate peroxidase (APX), glutathione reductase 
(GR) (Fig. 7), and catalase were differentially up-
 
Fig. 6. Time-course changes in the activity and 
transcript of FeSOD (a-c) and MnSOD (d, e) of 
U. fasciata in response to 30%  (open circle) or 
90%  (closed circle). Data are means ± SD (n=3) 
and asterisk indicates significant difference by 
t-test (Sung et al. 2009).
    U/va fasciata underwent oxidative stress in response 
to hypersaline condition (Lu et al. 2006; Sung et al. 
2009). To counteract oxidative stress, antioxidant 
enzymes including manganese superoxide dismutase 
(MnSOD), FeSOD, APX, GR, and catalase were 
150: 1-15.) noted that hypersalinity stress increased 
antioxidant defense enzyme activity. Therefore, we 
nology 11: 199-209).
hydrogen peroxide regulation of antioxidant 
defense system in Ulva fasciata. Marine Biology 
 
have determined the expression of genes of these 
antioxidant enzymes in U. fasciata in response to 
hyper-salinity stress (Sung et al., (2009) Hypers-
alinity and hydrogen peroxide up-regulation of 
gene expression of antioxidant enzymes in Ulva 
fasciata against oxidative stress. Marine Biotech-
119
up-regulated by short-term (Sung et al. 2009) or 
long-term (Lu et al. 2006) hypersalinity (90% ) 
treatments. However, the capacity for scavenging 
ROS was not sufficient for inhibiting ROS over-
production, as reflected by a significant H2O2 
accumulation after exposure to 90%  . As a result, 
macromolecules including membrane lipids, 
proteins, and nucleic acids are potentially oxidized. 
Therefore, in addition to the activation of antiox-
idant defense system, the removal and repairing of 
oxidative macromolecules, such as oxidatively 
damaged proteins, and the modification of other 
metabolic changes have become critical for the 
adaptation of U. fasciata to hypersalinity-induced 
 oxidative stress.
Fig. 7. Time-course changes in the activity and 
transcript of APX (a,b) and GR (c,d) of U. 
fasciata in response to 30%  (open circle) or 
90%  (closed circle). Data are means ± SD (n=3) 
and asterisk indicates significant difference by 
t-test (Sung et al. 2009).
5. Conclusion
high salinity conditions. The molecular acclimation 
signals and the following network in sensing the 
outer salinity changes are now developed.
References
Kirst GO. 1990. Salinity tolerance of eukaryotic 
          marine   algae.   Annual   Review   of   Plant 
          Physiology and Plant Molecular Biology 40: 
          21-53
Lee TM,  Liu  CH.  1999.  Regulation  of  NaCl-
          induced proline accumulation by calmodulin 
          via a modification of proline dehydrogenase 
          activity  in  Ulva  fasciata  (Chlorophyta). 
          Australian Journal of Plant Physiology 26: 
         595-600
Lee TM, Liu CH. 1999. Correlation of decreased 
         calcium contents with proline accumulation 
          in the marine green macroalga Ulva fasciata 
          exposed   to   elevated   NaCl   contents   in 
          seawater. Journal of Experimental Botany 
         50: 1855-1859
Lu IF, Sung MS, Lee TM. 2006. Salinity stress 
          and hydrogen peroxide regulation of antiox-
          idant defense system in Ulva fasciata. Marine 
          Biology 150: 1-15.
Sung  MS,  Hsu  YT,  Hsu YT,  Wu TM,  Lee TM. 
         2009. Hypersalinity and hydrogen peroxide 
         upregulation of gene expression of antioxidant 
         enzymes in Ulva fasciata against oxidative 
          stress. Marine Biotechnology 11: 199-209
   
  Ulva fasciata modulates gene expression, 
including protein destination and synthesis, the 
antioxidative defense system, and the repairing 
system of oxidized proteins, for the acclimation to 
 
of intertidal green macroalga Ulva fasciata Delile 
to high salinity stress was examined by the constr-
uction of a forward cDNA library via the suppre-
ssive subtractive hybridization between 30%   and 
90%  (24 h) and by the time course dynamics of 
several abundantly expressed genes. Among the 
genes with known sequences, the expressed sequ-
ence tags are abundant in the function of protein s
ynthesis (ribosomal protein) and destination. The 
Tse-Min Lee, Tsure-Meng Wu, Ming-Shiuan Sung et al.


Ulva as a Model for the Study of Environmental stress in Intertidal Macroalgae

玉木, 尚之

音楽と感情　-「声無哀楽論」の「秦客」の論-

池ノ内, 千乃

― 62 ―
入院患者における転倒・転落防止
－エビデンスに基づくアセスメント・スコアシートの作成を目指して－
　　　　　　　　　　　　　　　　　　　　転倒・転落防止対策チーム
　　　　　　　　　　　　　　　　　　　　　　池ノ内 千乃
キーワード：転倒･転落、アセスメントスコア、入院時アセスメント
Ⅰ．はじめに
　我々が従来から使用している転倒・転落アセスメント項目は、転倒・転落のリスクを広範囲にとらえ、
詳細に評価しようとしている。そのため、多数の調査項目を取り入れた詳細なスコアシートが普及してい
るが、その有用性については明らかなエビデンスに欠ける項目や、客観的評価が困難な項目も含まれてい
る。そこで、平成 19 年度木村看護教育振興財団の看護助成を受け、転倒・転落防止のための入院時アセ
スメントにおいて、臨床現場での使用に適した評価項目の選定を目的として検討を行ったものを報告する。
Ⅱ．研究方法
　１．対象者
　　　平成 18 年 11 月 1 日～ 11 月 30 日にＡ病院に入院した全患者 652 名
　２．調査期間
　　　平成 18 年 11 月～平成 19 年 2 月
　３．データ収集方法
　　　すでに臨床で使用されている、①年齢、②既往症、③感覚、④環境変化、⑤機能障害、⑥活動領域、
⑦認識力、⑧薬剤、⑨排泄、⑩症状、⑪履物、⑫その他の 12 主要項目に含まれる全 64 項目からなる「転
倒・転落アセスメント・スコアシート」を用いて、病棟看護師にアセスメントしてもらう。アセスメン
トの時期は入院時および、入院中に転倒・転落が生じた場合には転倒・転落イベント直前の評価を加え
ることとした。
　４．データ分析方法
　　　入院時の危険度スコアを評価し、その後入院中に転倒・転落イベントのあった患者の各評価項目につ
いてカイ２乗検定を行い、有用な項目の選定を行う。
　５．倫理的配慮
　　　当院規定の倫理的配慮を行い承認された。
Ⅲ．結　　果
　１．患者の属性
　対象患者 652 名の平均年齢（ｍ± SD）は 59.0（± 21.7）歳、その内、転倒者は 16 名で平均年齢は 65.5（±
19.3）歳であった。
　２．転倒・転落アセスメント・スコアシートの 12 主要項目に含まれる全 64 項目について転倒なし群と転
倒あり群でカイ２乗検定を行った結果、入院時転倒・転落アセスメントに有用な 14 項目を抽出した。
①転倒したことがある　②聴力障害がある　③足腰の弱り、筋力の低下がある　④車椅子・杖・歩行器
を使用している　⑤移動に介助が必要である　⑥ふらつき・失調性歩行がある　⑦付属物の装着（点滴・
吸引器・ドレナージ以外）　⑧混乱がある　⑨判断力、理解力の低下がある⑩不穏行動がある　⑪トイ
レ介助が必要（ポータブルトイレ使用を含む）　⑫浮腫がある　⑬説明しても守らない患者　⑭スリッ
パを履いている
― 63 ―
Ⅵ．今後の展望
　今回選定された項目は、入院時の転倒・転落アセスメント項目であり、今後は入院後に刻々と変化する
患者の状態に合わせたアセスメント項目を追加する事が必要であると考える。我々は、より効果的で簡便
な項目選定を目指し現在も検討を重ねている。
平成 23 年 8 月 21 日　第 9回日本医療マネジメント学会高知県支部学術集会（高知）にて発表〔 〕


入院患者における転倒・転落防止 －エビデンスに基づくアセスメント・スコアシートの作成を目指して－

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Studies of Cooperation between Schools and their Regions in Sports Activities in Schools ~For Elementary, Junior High, Senior High Schools in Kochi Prefecture~

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