Journal of Life Sciences Volume 7 Number (6)
J LS
Journal of Life Sciences
Volume 7, Number 1, January 2013 (Serial Number 57)
Contents
Cell and Biochemistry
1 The Overexpressed FAK (Focal Adhesion Kinase) in Higher Grade Human Urothelial Tumors
Baz Ahsene, Ousmaal Mohamed El Fadel, Mammeri Saâdia, Zineddine-Charef Amir, Frederic Boudard, Frederic Hollande, Belal Tahar and Jean Giaimis
8 Features of Structurization at Participation of Guanidine Groups of Arginine in Life Cycle in Population of E. coli
Tropynina Tatyana, Ivanova Evilina, Vafina Gulnara and Ivanov Ruslan
13 Effects of Transportation Stress during the Hot-Dry Season on Some Haematological and Physiological Parameters in Moroccan Dromedary Camels (Camelus dromedarius)
Mohammed El Khasmi, Youssef Chakir, Fouad Riad, Abdallah Safwate, El Hassane Tahri, Mohamed Farh, Najia El Abbadi, Rachid Abouhafs and Bernard Faye
Ecology and Environment
26 Effect of Limiting Access to Drinking Water on Carcass Characteristics, Meat Quality and Kidneys of Rabbits
Ben Rayana Aniss, Ben Hamouda Mohamed, Kaddech Anis, Amara Abdelkader and Bergaoui Ridha
31 Reproductive Strategies of a Terrestrial Snail along an Altitudinal Gradient on an Oceanic Island
Ana Filipa Ferreira, António Manuel de Frias Martins, Regina Tristão da Cunha, Paulo Jorge Melo and Armindo dos Santos Rodrigues
42 Bluetongue: A Hypothesis of Control Strategy through Decrease of Culicoides and Their Associated Damage in Farm
Rolesu Sandro, Aloi Daniela, Cappai Stefano, Mereu Piras Pierpaola, Fois Francesco, Satta Giuseppe, Palmas Costantino, Ecca Anna Rita and Pulina Giuseppe
51 Removal of Different Dyes by Pseudomonas fluorescens
Sewgil Saaduldeen Anwer and Sawan Merkhan
57 Preparation of Organic Selenocystine Using Locally Isolated Bread Yeast
Fouad Houssein Kamel
63 To a Question about Forecasting Number of Micromammalia (Rodentia)
Nadezhda Antonets, Aleksandr Balalayev and Мargarita Shumkova
Interdisciplinary Researches
69 A Prospective Approach for Exploring Potential Biofuel Plants
Shubo Zhang, Jing Liu, Xiaobai Jin, Daniel Wai Tin Chan and Miao He
76 Search of Salmonella in Meat and Dairy Products
Sonia Khosrof, Sbei Rim and Wejden Suihi
84 An Advanced Catch-and-Release Trap for Controlling the Red Palm Weevil
Nabawy Metwaly
89 Tracking, Obedience and Defense between Belgian Shepherd Malinois and German Shepherd Dog
Ivana Gardiánová, Martina Helclová, Stanislav Koráb and Lenka Hradecká
92 Evaluation of Road Accidents in Pristina in the Period 2009-2012
Basri Lenjani, Salih Krasniqi, Nehat Baftiu, Ilaz Bunjaku and Arianit Jakupi
Jan. 2013, Vol. 7, No. 1, pp. 1-7 Journal of Life Sciences, ISSN 1934-7391, USA
The Overexpressed FAK (Focal Adhesion Kinase) in Higher Grade Human Urothelial Tumors
Baz Ahsene 1 , Ousmaal Mohamed El Fadel 1 , Mammeri Saâdia 2 , Zineddine-Charef Amir 3 , Frederic Boudard 4 ,
4 5 Frederic Hollande 5 , Belal Tahar and Jean Giaimis 1. Laboratory of animal physiology and cell signaling, ENS Kouba, Algiers 16000, Algeria
2. Hospital Beni Messous, Algiers 16000, Algeria 3. Hospital Mustapha, Algiers 16000, Algeria
4. UMR Qualisud- Faculty of Pharmacy, University of Montpellier I, Montpellier 34093, France 5. Laboratory-body and structure of matter, ENS Kouba, Algiers 16000, Algeria
Received: July 13, 2012 / Accepted: September 17, 2012 / Published: January 30, 2013.
Abstract: Malignant transformation of normal cells involves important structural and functional changes, particularly in cell adhesion. In this study, we wanted to assess whether changes in the expression of FAK, a tyrosine kinase, which is recruited to focal adhesions and plays a key role in cell migration, proliferation and survival, could reflect the invasive capacity of bladder carcinomas. The aim of this study was to evaluate the FAK expression in cancer cells as an important prognostic factor of the evolution of bladder carcinomas. Tumor and paired peritumoral biopsies were obtained during transurethral endoscopic resection or cystectomy of bladder tumors in 280 patients at the Urology Unit of the Mustapha Hospital of Algiers and the Hospital of Tizi-Ouzou (Algeria). The authors studied FAK expression in samples from bladder carcinomas at different stages of malignant transformation by western blot analysis using a specific anti-FAK antibody. Western blot is one of the most common laboratory techniques; it is used to detect the presence of a specific protein in a complex mixture extracted from cells. A weak increase in FAK expression was observed in tumors of grade
1 and 2 (1.65; 2.99) as compared to healthy tissues; it became particularly important in grade 3 tumors; the authors show that FAK levels significantly increased gradually according to the tumor stage.
Key words: Bladder, cancer, focal adhesion kinase, retrodifferentiation.
1. Introduction their predicting value are currently major research goals. At present, no single tumor marker has real
Tumors of the urinary tract are frequent and very predictive value in the case of infiltrating cancer of the often malignant. Although, preservation of the organ bladder [2]. Cell adhesion proteins play a critical role could be achieved in certain situations, cystectomy is in tumorigenesis. As they ensure the coupling between nevertheless regarded as the standard treatment in the extracellular matrix and actin cytoskeleton, changes in case of bladder cancer [1]. In spite of the progress of their expression can lead to modifications in focal the surgical techniques and the introduction of adhesions that facilitate the diffusion of factors protocols of systemic chemotherapy, many patients involved in tumor progression and metastasis with tumors infiltrating the bladder often die within formation [3]. A major component of focal adhesions few years from the initial diagnosis. Therefore, the is FAK (focal adhesion kinase), a tyrosine kinase identification of tumor markers and the evaluation of which is recruited to focal adhesions transmits signals
Corresponding author: Baz Ahsene, Ph.D., professor, from the extracellular matrix to the cytoplasm through
research fields: animal physiology and cell signaling. E-mail: integrin-dependent signaling pathways. It is [email protected].
2 The Overexpressed FAK (Focal Adhesion Kinase) in Higher Grade Human Urothelial Tumors
implicated in many biological processes, such as
2. Materials and Methods
proliferation, cell motility and survival as well as in Tumor and paired peritumoral biopsies were the malignant transformation of normal cells [4, 5]. In obtained during transurethral endoscopic resection or cancer cell lines, FAK inhibits apoptosis by activating cystectomy of bladder tumors in 280 patients at the the PI3-kinase (phosphatidylinositol 3’-OH-kinase)— Urology Unit of the Mustapha Hospital of Algiers Akt/PKB (protein kinase B) survival pathway and and the Hospital of Tizi-Ouzou (Algeria). The ultimately by inhibiting the caspase-3 cascade [6, 7]. following informations were recovered: age, weight, FAK, PKB and Src are part of a complex network of gender, tumor antecedents, treatments, tumor intracellular signals involved in cancer cell regulation description (aspect, size, and number of tumor [8]. Conversely, inhibition of FAK expression in nodules and presence or not of metastasis). Patients
different cancer cells makes them loose their were informed about this research and gave their
attachment and causes apoptosis [9, 10]. In lung tumor
written agreement.
cells treated with anti-GD2 ganglioside antibodies, One half part of each biopsy was used for the
apotosis is elicited by reducing FAK phosphorylation
and activating p38 [11]. Cleavage of FAK by Apo-2L histological analysis and the other half part for protein or Fas can induce morphological changes analysis. For histology, tissues were rapidly fixed, characteristic of apoptotic cells (detachment from the
dehydrated, embedded in paraffin, and 2 µm to 5 µm substratum and loss of cell-cell interaction) in Jurkat T
serial sections were obtained using a Minot microtome. and HL60 cells [12]. In conclusion, FAK deregulation
Sections were then stained with hematoxylin-eosin might play a role in cancer development and
and observed under a Zeiss Axioskop-40 microscope progression [13, 14] and elevated expression of FAK
and microphotographs were taken using an Axiocam in human tutors has been correlated with increased
digital camera. Determination of the histological type, malignancy and invasiveness [15, 16]. Bladder stage and rank was carried out according to the WHO cancers constitute a major public health preoccupation
classification.
in Algeria. Men are more affected (79.64%) than
2.1 Antibodies
women (20.36%) and especially older people as bladder cancers are particularly frequent between 50
The antibodies used in this study were: mouse and 80 years. The recurrence percentage of urothelial
anti-FAK monoclonal antibody, clone-77 (1: 1000) carcinomas is 29.82% in women and 21.52% in men.
(Transduction Laboratories, Lexington, KY, USA), Since their diagnosis is difficult and often late, new
mouse anti-actin monoclonal antibody, clone AC-40 diagnostic and prognostic tools are particularly needed.
(1: 1000) (Sigma, St Louis, Missouri, USA), and Therefore, we asked whether changes in FAK
mouse IgG secondary antibody coupled to HRP expression level in bladder tumors at different stages
(Horseradish peroxidase) (1: 5000) (International of dedifferentiation could reflect tumor evolution. To
Chemicon, Inc.).
this aim, biopsies of bladder tumors of patients
2.2 Total Protein Extraction
submitted to transurethral resection or cystectomy were recovered immediately after surgical extraction.
Sample from each biopsy was weighed and then After staging of the tumors accoroding to the WHO
finely powdered in a mortar containing some liquid classification [17], FAK expression was evaluated
nitrogen. Powdered tissues were suspended in RIPA with an anti-FAK specific antibody. Our results show
lysis buffer (1 M Tris pH 7.5, 5 M) NaCl, 5% Triton that FAK was overexpressed in bladder cancers as
X-100, 5% SDS (sodium desoxycholate), 100 mm compared to normal bladder tissue.
sodium vanadate, 1 M DTT, phosphatase inhibitor
3 cocktail, protease inhibitor cocktail (Sigma), vortexed
The Overexpressed FAK (Focal Adhesion Kinase) in Higher Grade Human Urothelial Tumors
For western blot analysis, separated proteins were and centrifuged at 4 °C at 14,000 g for 10 min.
transferred onto nitrocellulose transfer membranes Supernatants were stored at +4 °C; one aliquot was
(ProteanR BioScience) at 3.5 mA/cm 2 intensity with a used for measuring protein concentration according to
15 V constant voltage for 75 min. After protein the Bradford method [18] with a spectrophotometer
transfer, membranes were rinsed in PBS-0.1% Tween (UV-1201, Shimadzu) at a wavelength of 595 nm; the
20 for 2 min, blocked with PBS-0.1% Tween 20-milk rest was kept for western blot analysis.
3% (v/v) for 30 min, and finally rinsed quickly with PBS-0.1% Tween 20 (v/v) for 2 min. Membranes were
2.3 SDS-PAGE and Western Blotting then incubated with the primary antibodies (anti-FAK
Lysates that were denatured at 100 °C for 4 min and or anti-Actin) in PBS-0.1% Tween 20-milk 3% (v/v)
15 µL (1 µg/µL proteins) were loaded on 8% overnight at 4 °C, rinsed once with PBS-0.1% Tween SDS-PAGE gel [19] and separated at 20 mA/gel to
20 (v/v) and then washed once for 15 min and twice 200 V. Gels were then either stained with Coomassie
for 5 min, and incubated with mouse IgG coupled to blue or processed for western blot analysis. For
HRP at room temperature for 1 h After washes in Coomassie blue staining, gels were transferred in 250
PBS-0.1% Tween 20 as previously, antibody binding mg of Coomassie Shining Blue R250 in 90 mL of
was visualized with ECL (electrogenerated 50:50 methanol:distilled water (volume/volume) and
chemiluminescence) detection reagents (Amersham)
10 mL of acetic acid for 12 h maximum. Gels were and bands from autoradiographic films were analyzed
with the Photo-Capt MW system and Image J software. for 4 h maximum. After then, gels were photographed
then washed in methanol/acetic acid/H 2 O four times
FAK expression values were then corrected to Actin
Fig. 1 Protein profiles of normal and tumoral bladder tissues on SDS-PAGE.
4 The Overexpressed FAK (Focal Adhesion Kinase) in Higher Grade Human Urothelial Tumors
expression (by dividing the FAK density by the actin massive form; only 3% corresponded to correction index) (Fig. 2). The tumor/normal tissue
adenocarcinomas. Following the TNM ratio was then calculated by using the Image J
(tumor-node-metastasis) system, tumors were Software.
classified as follows: pTa (12%), pT1 (49%), pT2 (28%), pT3 (5%) and pT4 (6%). In situ carcinomas
2.4 Statistics were very rare and accounted for only 1% of all
Values of FAK expression were compared using tumors. Moreover, grading according to the tumor cell unpaired Fisher’s t-test (significant at P < 0.05; *: P <
differentiation showed a clear predominance of grade 0.05; **: P < 0.01; ***: P < 0.001).
2 tumors (Fig. 4); all the tumors excised by cystectomy or cysto-prostatectomy were grade 2 or 3.
3. Results
The urinary bladder of paired healthy tissues
3.1 Clinical Observations and Histological showed a transitional epithelium made of 6-7 layers of
Classification regular urothelial cells resting on the basement membrane that separates the epithelium from the
Bladder tumors were excised mainly by connective tissue (lamina propria), the muscularis transurethral endoscopic resection (80% of the and adventitia (Fig. 5a). In grade 1 tumors, an patients) or by cystectomy (20%). The following increased number of cellular layers in the transitional clinical signs were observed (in order of importance): epithelium were found. Tumor cells were characterized hematuria, frank cystitis, dysuria, pollakiuria as well
as abdominal and pelvic pain. Macroscopically, the carcinomas generally
presented a budding form (76.66% of patients), sometimes pedunculate (13.33% of patients) and seldom sessile (6.66%) or papillary. There was a marked predominance of multiple tumors (70% of the
Percentages (%
patients). Tumors were often localized in the left and right lateral walls as well as in the trigonal area of the bladder (Fig. 3).
Pathological analysis of the tumors indicated that
97% were urothelial carcinomas in their papillary or Fig. 3 Frequency of bladder tumors in all patients (n = 280).
Percentages (%
Fig. 2 Western blot analysis of FAK during urothelial cell retro-differenciation.
Fig. 4 Tumor localization in the bladder.
5 by anisocytosis, anisocaryosis and rare mitosis; cell
The Overexpressed FAK (Focal Adhesion Kinase) in Higher Grade Human Urothelial Tumors
Specifically, a weak increase in FAK expression was polarity was still preserved (Fig. 5b). In grade 2
observed in tumors of grade 1 and 2 (1.65; 2.99) tumors, the transitional epithelium was very thick,
(significant for grade 2) as compared to healthy tissues; with nuclear anomalies; cells were bulkier with
it became particularly important in grade 3 tumors [16, increased mitotic activity and moderate disturbances
18]. In addition, the total protein profiles of of cell polarity, which gave to the epithelium a
samples from healthy tissues and grade 1, 2 and 3 somewhat disorganized aspect (Fig. 5c). In grade 3
tumors revealed increased staining of a 125 KDa m.w. tumors, signs of dedifferentiation were obvious, with
band which could correspond, beside other proteins, to cytonuclear atypia and architectural disorganization of
the FAK protein.
the transitional epithelium. Adenocarcinomas, which were rarely found, were organized in glands or tubules
(Fig. 5d).
3.2 FAK Expression Increases with the Degree of Tumor Malignancy
The authors then evaluated whether changes in FAK expression between healthy and tumor tissues, and among tumors of different grade, are found. Indeed, whereas in healthy tissues FAK could barely
Fig. 6 Quantification of FAK expression in urothelial
be detected, its expression increased concomitantly tumors. *: P < 0.05; **: P < 0.01; ***: P < 0.001 and no
with the grade of the tumor (Figs. 2 and 6).
significant (NS) at P > 0.05.
Fig. 5 Haematoxylin-eosin staining of healthy and tumoral bladder tissues; Transversal section of normal bladder: epithelium (E), chorion with connective tissue (TC), muscularis (M) (HE, 100 ×); Non infiltrative papillary urothelial tumor grade1: the epithelium has more than six layers; basal lamina (LB) is intact; connective and vascular axis are thin (AC); few atypical nuclei, and rare mitosis (TRI, 1000 ×); Urothelial carcinoma of grade 2 infiltrating the chorion (stage pT1a): basal lamina LB is destroyed; many blood vessels (VS) are observed; chorion was infiltrated (I) by tumor lobules (L) (PM, 400 ×); At higher magnification, an increased cellular density with abnormal mitosis (M), and polymorphism characterized by marked anisocytosis and anisocaryosis can be seen (HE, 1000 ×).
6 The Overexpressed FAK (Focal Adhesion Kinase) in Higher Grade Human Urothelial Tumors
4. Discussion
tamoxifene suppressed chemically-induced tumor formation. Moreover, loss of Fak inhibited malignant
The incidence of bladder cancer is increasing in the progression of already formed benign tumors; loss of
industrialized countries and particularly in Western Fak was also associated to a reduction of in vitro
Europe [20]. Currently, the prognosis of bladder migration and with keratinocytes apoptosis both in
tumors is based on anatomo-clinical criteria; however, vitro and in vivo [29]. Furthermore, high levels of
molecular and genetic analysis could allow an FAK were correlated with migration of cancer cells,
identification of markers whose expression is related invasion, and ability to metastasize [30].
to the tumor stage and grade. Such markers could help The authors’ study, by associating clinical,
to identify patients at risk and to predict their response histological and biochemical data, highlights the to different therapeutic protocols.
implication of FAK in the development of malignant The aim of this work was to assess whether changes
features in human bladder carcinomas in vivo. These in FAK expression could play a potential role in the
results are in agreement with previous work carried development and progression of bladder tumors in
out in other type of cancers [8, 23, 30-32]. vivo. Indeed, we show that increased FAK expression
is associated with tumors of increasing malignancy
5. Conclusion
and with higher invasive potential. Proteins of the The development of new tools targeting critical cellular junctions are involved in the maintenance of
molecular anomalies involved in tumor progression is the architecture and the polarity of epithelial cells [21].
very much needed. Indeed, the rational use of tumor Loss of adhesion due to deregulation of factors
markers may allow the choice of individualized localized at focal adhesion sites, such as FAK, can
therapeutic strategies for each patient affected by facilitate migration of tumor cells and therefore
bladder cancer. However, currently, many markers are formation of metastasis [22]. Indeed, increased associated with tumors with different staging and
phosphorylation of FAK favors metastatic invasion of grading; to be really useful, these markers must be colon cancer cells [23, 24]. Overexpression and/or
validated by multicentric prospective studies. activation of Src and FAK increase cellular
Acknowledgments
proliferation, survival and metastasis [25, 26]. FAK is involved in complex regulatory pathways, implying
The authors would like to thank Prof. Jacques adhesion with the extracellular matrix, but also growth
Mercier (Montpellier, France) and Prof. Jean Pierre factors, hormones and chemokines [27]. For instance,
Bali (Montpellier, France) for helpful comments. This interactions between FAK and Src or Bcr/Abl have
project was supported by DAHR (the Development been described [13]; in Src-induced colon cancer cells
Agency of Health Research), Oran (Algeria) and by EPS8, FAK expression was deregulated through
the Ministry of Research of Algeria. mTOR and STAT3, both at the level of gene
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Jan. 2013, Vol. 7, No. 1, pp. 8-12 Journal of Life Sciences, ISSN 1934-7391, USA
Features of Structurization at Participation of Guanidine Groups of Arginine in Life Cycle in Population of E. coli
Tropynina Tatyana, Ivanova Evilina, Vafina Gulnara and Ivanov Ruslan Institute of Biology, Ufa Science Centre, Russian Academy of Science, Ufa 450054, Bashkortostan, Russia
Received: September 12, 2012 / Accepted: November 14, 2012 / Published: January 30, 2013.
Abstract: The purpose of the given work was the experimental analysis of features of Arg-X proteolysis in proteom of supramolecular structures of bacterial cells during their life cycle. The basic attention was devoted to relaxation of Arg-X sites of proteom in association with the evolutionary significance of Arg-rich histones in the eukaryotic kingdom. These properties were not studied in the prokaryotes. Cells of E. coli were grown to the stationary phase, collected by centrifugation and washed. All cells were taken over from 50 min to 430 min at intervals of 20 min and were preserved in glycerol. The supramolecular structures were fractionated from bacterial cells by increasing ionic strength of solution. The Arg-X activity was assessed by cleavage of Arg-X bonds in the arginine-enriched protein protamine in all cell fractions. We have shown that during the stationary phase in the life cycle of E. coli, there are a high continuous activity of the Arg-X processing at the level of “cytoskeleton” of the cell and bright cyclic activity in the cytoplasm.
Key words: Arginine, Arg-X protease-sensitive, supramolecular structures, nucleoid, E. coli.
1. Introduction eu- and prokaryotic organisms can have the common features. It is assumed that Arg-X proteolysis
The main components in the structural organization functions are in a chromatin of eukaryotic cells. of DNA in the eukaryotic cells are histones. The Probably the Arg-X proteolysis functions in a bacterial special evolutionary conservatism of amino acids chromosome taking an active part in reorganization sequences of histones enriched by arginine in the
structure of nucleoid.
eukaryotic kingdom is marked. Extremely reactive The purpose of the given work was the guanidine group in δ-position is structural feature of experimental analysis of features of Arg-X proteolysis arginine. Guanidine group of arginine possesses the in proteom of supramolecular structures of bacterial strongest basic properties in comparison with lysine.
cells during their life cycle.
This group actively is exposed to various sorts of modifications which lead to change of interrelations of
2. Materials and Methods
supramolecular complexes in process onto- and
E. coli strain JC-158 [1], used in the work, was philogenesis of eukaryotes. As to bacterial cells, E.
kindly provided by colleagues I.V. Stupak and E.E. coli is investigated most full. Stupak. Cells of E. coli were grown in rich medium It is proved that the histone-like proteins is formed LB (Luria Bertani) [2] to the stationary phase, the structure of nucleoid bacteria. Similarity between collected by centrifugation and washed with Tris histone-like proteins of prokaryotes and histone buffer. The first sample was taken in 50 min after the proteins of eukaryotes testifies that packing of DNA at start of incubation. All cells were collected over from
50 min to 430 min at intervals of 20 min, and
Corresponding author: Ivanova Evilina, Ph.D., research field: biochemistry. E-mail: [email protected].
preserved in glycerol by the method [3]. The
Features of Structurization at Participation of Guanidine Groups
of Arginine in Life Cycle in Population of E. coli
supramolecular structures were fractionated from amino acids: 22-Arg, 4 Ser, 3 Pro, 2 Gly and 2 Val. bacterial cells according to the method [4]. Proteome
Activity Arg-X proteolysis was expressed in nM of structures from E. coli were fractionated by breaking
arginine/( μ protein·s). The numbers and points on the of the weak and strong bonds of supramolecular
graphs represent the arithmetic mean data. structures. It was done by increasing ionic strength of
3. Results and Discussion
solution: 0.14 M NaCl; 0.35 M NaCl; 2 M NaCl; 6 M guanidine-hydrochloride with 0.004% 3.1 Physiology-Biochemical State of E. coli Cells β-mercaptoethanol at 0.01 M Tris-HCl buffer (pH 6.8).
The understanding of biological processes is Extraction of proteins by increasing the ionic strength reached much more easily by means of modeling of the salt gradient, leading to a weakening of the organisms. In a bacterial cell, the chromosome is electrostatic interaction between proteins and packed in the form of the compact structure connected adsorbent, are the usual methods of protein chemistry with a membrane. This DNA-membrane complex
[5]. The fraction of Cp (cytoplasm) was isolated by provides structural packing of a chromosome,
0.14 M NaCl. The fraction of supramolecular structure of loosely bound with cellular rest (Sp I) was received
replication and segregation [7].
by the extraction of 0.35 M NaCl. The fraction of In an active growth phase (the period from 50 min supramolecular structure of tightly bound with cellular
to 190 min) cells of bacteria grow with the highest rest (Sp II) was isolated by 2 М NaCl. CR (Cellular
speed in our experiment (Fig. 1). At gradual depletion rest) was obtained by extraction 6
МGu·HCL of necessary nutrients and accumulation of products of (guanidine hydrochloride) and 0.004% metabolism, the growth rate of bacteria is decreasing
β-mercaptoethanol [4, 6]. The amount of protein in the (from 190 min to 330 min—delay phase). Then supramolecular structures was determined by growth of bacteria is stopping (from 330 min to 430 Bradford in our modification [4]. Arg-X activity was
min), the culture is passing into a stationary phase. It valuated from digestion of Arg-X bonds in the
is believed that when the culture of bacteria pass to a protamine Salmine-AI (Merk, Darmstadt, Germany), a
stationary phase the differentiation program is started, protein enriched by Arg. This protein comprises 33
leading to that cells become metabolic less active and
eluat mL
Amount of cells in 1.5
Growth Phases
Fig. 1 Dynamics of increasing the population density of cells E. coli in the life cycle.
10 Features of Structurization at Participation of Guanidine Groups of Arginine in Life Cycle in Population of E. coli
more resistant to stressful factors [8]. Many functions stressful influences testifies that in the specified are induced upon transition of culture to the stationary
conditions in cells there is a raised expression of the phase. And it’s activated at the limitation of nutrients
genes necessary for protection [8]. In the conditions of [8]. Under these conditions, the expression of most
a stress microorganisms become hardier and, their bacterial genes is significantly reduced. However,
pathogenic factor is increasing [9].
there is an induction of an expression of a large
3.2 Arg-X Protease-Sensitive in Supramolecular number of other genes and synthesis of specific
Structures of E. coli Cells
proteins, first of all what provide stability of bacteria to various adverse conditions is stimulated [8]. Thus
Studying of thin molecular and supramolecular life cycle of bacteria includes the periods of the active
structures is capable to deepen our understanding of growth alternating with the periods of delay and the
the spatial-temporary and functional organization of a termination of growth (Fig. 1). In natural conditions,
prokaryotic cell. As a matter of fact, cellular level of bacteria are seldom lived in abundance conditions,
the organization of life activity of a bacterium can be allowing exponential growth [8]. Short periods of
considered from a position of well organized rapid growth are alternating with long periods of
“associate” of molecules, the supramolecular starvation (stationary phase); the cells are undergoing
structures which are constantly cooperating with each various unfavorable factors for their life activity.
other and environment. In Albrecht-Buhler’s opinion Inthese conditions the bacteria should be lived for a
[10], supramolecular descriptions of form-operating long time. And then come back to an exponential
processes are valuable as interactions of many phase when influence of starvation and other adverse
molecules are integrated into them because signals effects will be removed [8].
along their linear structures at the expense of This period also is characterized by activation of
association and a dissociation of molecules can move. molecular mechanisms of adaptation of We discuss results of experiment in Fig. 2 from these microorganisms to a stress. The fact that cells in the
positions. Fig. 2 shows the characteristics stationary phase are essentially steadier against of spatial-temporal Arg-X protease—sensitivity of.
ll
μ protein) on ce ·
g./(s
Growth Phases
Fig. 2 Arg-X proteolysis in the supramolecular structures (cell fractions) of E. coli during life cycle: cytoplasm (Cp); loosely bound supramolecular structures with CR (Sp I); tightly bound supramolecular structures with CR (Sp II); cell rest (CR).
Features of Structurization at Participation of Guanidine Groups
of Arginine in Life Cycle in Population of E. coli
supramolecular structures of bacterial cells of E. coli. genes of bacteria in these conditions. Nucleoid The data was shown that in the period of transition to becomes more compact owing to change of structure
stationary growth phase (phase of active stress) in the and the quantitative contents of the proteins bonded cytoplasm was increased cyclic splash of Arg-X
with DNA. Modulation of nucleoid plays the essential proteolysis. A number of studies have shown that in
role in DNA preservation in the cells being in the stationary growth phase the qualitative and
conditions of starvation and exhaustion of a source of quantitative changes in protein composition of energy, and also repressions of the majority of nucleoid influence on DNA compaction [8]. At delay
bacterial genes. It was shown that bacterial nucleoid and termination of growth, the cell culture of E. coli
was being extracted experimentally at presence 1 М undergoes significant morphological changes. Cell
NaCl, and Arg-X protease-sensitivity was being shape is changing: if during the period of rapid growth
localized in supramolecular structures of cytoplasm of of the culture, they have a rod-shaped form, at the
stationary growth phase (320, 390-410 min) of E. coli delay of growth of the culture, they become much
(Fig. 2). It was assumed that during this period, smaller and almost spherical. It is a consequence of
essential changes in a remodeling and regulation of several cell divisions without an increase in cell mass initiation of translation system were being carried out.
It is believed that the sharp decrease in cell size can Because it is known that system of transcription and contribute to the survival of bacteria by increasing
translation is functioning together in cytoplasm of their number. The cytoplasm of cells is condensing;
bacterial cell. In a number of studies, essential the volume of the periplasm is increasing [8]. The rate
distinctions at the stage of a termination of translation of proteolysis of the cellular proteins is also increasing
at eukaryotes and prokaryotes are noted [11]. It is also by at least several times during starvation of cells in
shown in Fig. 2 (310-430 min) that transition of the stationary phase of growth. The amino acids
bacterial cells to the stationary phase is accompanied released as a result proteolysis of proteins are used for
by demonstration of weak, but continuous Arg-X synthesis of new proteins [8]. It is quite possible that
proteolysis at level of supramolecular structures of the during of this period biogenic amines are forming. For
CR (cellular rest). Apparently, the part of bacterial example, from arginine are forming agmatine. cells completely degrades. The question of what Agmatine in a certain dose is possessing poisonous
molecular mechanisms provide the program of properties. Global changes in gene expression at
differentiation of cells in the conditions of delay and transition of cells to a stationary growth phase occur at
termination of growth, activation (induction) of each stage of an expression of genes and include
expression of a large number of genes at decrease in changes in conformation of nucleoid, the apparatus of
the common level of an expression of bacterial genes transcription and translation. Chromosomal DNA of E.
remains insufficiently studied. Thus, at transition of coli is associated with 10 main types of structural
bacteria to the stationary phase, cells become proteins, forming nucleoid. These proteins are now
metabolic less active and steadier against stressor often referred to as nucleoid proteins; they play the
factors. In the conditions of stress at level of a important role in the regulation of such processes as
bacterial cell, there is the formation of free groups of replication, recombination and transcription which are
arginine, and arginine in turn also participates in required for cells [8]. At transition from the culture to
synthesis of biogenic amines, in particular agmatin.
a stationary phase, topological changes are occurring Our data showed that in proteom of genome of in chromosome of the starving cells, correlating with
bacterial cells the Arg-X system of proteolysis actively reduction of the general level of an expression of
functions in a stationary growth phase of bacteria.
12 Features of Structurization at Participation of Guanidine Groups of Arginine in Life Cycle in Population of E. coli
According to literature, it is known that at long action
References
of adverse factors, the increase in activity of
D.B. Myrphy, J.T. Pembroke, Transfer of the IncJ arginine-decarboxylase is observed. As a result of
plasmid R391 to recombination deficient Escherichia coli decarboxylation reaction free arginine turns in
K12: Evidence that R391 behaves as a conjugal biogenic amine-agmatin. In the conditions of the transposon, FEMS Microbiology Letters 134 (1995)
153-158.
stationary phase of life cycle of bacteria, the induction [2] T. Maniatis, E.F. Fritsch, J. Sambrook, Molecular of expression of the genes providing synthesis of
Cloning: A Laboratory Manual, Cold Spring Harbor proteins which are necessary for their stability to
Laboratory Press, Cold Spring Harbor, NY, 1982.
adverse conditions is being carried out. During this E.A. Ivanova, G.H. Vafina, Method of selection of plant cell nuclei, RF Patent, 1701747 B48 (1991).
period, there is a high continuous activity of the Arg-X
E.A. Ivanova, G.H. Vafina, A method of producing processing at the level of “cytoskeleton” of the
nuclear fractions with proteinase and inhibitory activity, cell and bright cyclic activity in the cytoplasm is
RF Patent, 1733471 B18 (1992).
[5] noted. R.K. Scopes, Protein Purification: Principle and Practice, Springer-Verlag, New York, NY, 1982.
4. Conclusion E.A. Ivanova, G.H. Vafina, T.S. Tropynina, A method of
producing fractions of E. coli cell with proteinase activity, It is known that in the conditions of a stationary
RF Patent, 2410428 B3 (2011). [7] S. Hiraga, T. Ogura, H. Niki, C. Ichinose, H. Mori,
phase of life cycle of bacteria, an induction of Positioning of replicated chromosomes in Escherichia
expression of the genes providing synthesis of coli, Journal of Bacteriology 172 (1990) 31-39. proteins which are necessary for their stability to
I.A. Khmel, Regulation of expression of bacterial genes adverse conditions is being carried out. During this
in the absence of active cell growth, Russian Journal of Genetics 41 (9) (2005) 968-984.
period high continuous activity of Arg-X of processing
A.G. Tkachenko, Bacteria and stress, Science of the Ural at level of “cytoskeleton” of the cell is noted. The
5 (2010) 4-5.
knowledge of mechanisms of regulation of expression
G. Albrecht-Buhler, In defense of “nonmolecular” cell of genes will allow finding ways of target biology, International Review of Cytology 120 (1990)
191-241.
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phases of cells necessary for nanotechnological eukaryotes and in prokaryotes is essentially different, designs.
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Jan. 2013, Vol. 7, No. 1, pp. 13-25 Journal of Life Sciences, ISSN 1934-7391, USA
Effects of Transportation Stress during the Hot-Dry Season on Some Haematological and Physiological Parameters in Moroccan Dromedary Camels (Camelus dromedarius)
1 1 1 1 1 Mohammed El Khasmi 1 , Youssef Chakir , Fouad Riad , Abdallah Safwate , El Hassane Tahri , Mohamed Farh ,
Najia El Abbadi 2 , Rachid Abouhafs 3 and Bernard Faye 4
1. Laboratory of Physiopathology and Molecular Genetics, Faculty of Sciences Ben M’Sik, University Hassan II-Mohammedia, Casablanca 20000, Morocco 2. Unit of Applied Radioimmunology, National Center for Energy Sciences and Techniques and Nuclear Techniques, Maamoura, Morocco 3. Prefectural Veterinary Service of Casablanca, Casablanca 20000, Morocco 4. CIRAD-ES, International Campus of Baillarguet, Montpellier 34398, Cedex 5, France
Received: August 28, 2012 / Accepted: November 19, 2012 / Published: January 30, 2013.
Abstract: The purpose of this study was to determine the effects of road transportation under heat conditions on some haematological [Ht (haematocrit), blood cells count and EOF (erythrocytes osmotic fragility)] and physiological [Tr (rectal temperature), HR (heart) and RR (respiratory rates), and circulating levels of Cor (cortisol), Glu (glucose) and minerals] parameters in Moroccan dromedary camels. The animals were subjected to road transportation stressor for 2 h by truck during the hot-dry season. Blood samples were collected before loading and transport, and at the end of transport. Transportation induced a significant increase (P < 0.05) of erythrocytes count, Ht, EOF, Tr, HR and RR by comparison to values observed before transportation. The same stress conditions induced a significant increase (P < 0.05) of plasma Cor (ng/mL) and blood Glu (mM) (220 ± 30 vs. 137 ± 20, 9.7 ± 1.2 vs.
6.4 ± 1.1 respectively) and a significant decrease (P < 0.05) of plasma magnesium (mM) (0.5 ± 0.1 vs. 0.9 ± 0.1) comparatively to pre-transportation values. These results indicate that road transportation associated to heat may be considered as a potent stressor which is able to induce several cellular alterations in camels. Further studies of an eventual protective role of vitamin C against haemolysis induced by transportation stress in camel are needed.
Key words: Cortisol, dromedary camel, glucose, haemolysis, hot-dry season, minerals, transportation stress.
1. Introduction Pituitary-Adrenal gland axis)) [1]. In dromedary camels, activation of HPAA by road transportation is
Road transportation of animals is a potent stressor, able to induce a significant release of Cor (cortisol) which results from vehicle motion, noise and vibration, and thyroid hormones [2, 3]. It is well documented and can change several of the animal’s physiological that transportation of livestock is a complex systems (e.g. cardiovascular, immune and endocrine multi-factorial and traumatic event [4] especially via the activation of HPAA (Hypothalamo- during the hot dry summer months characterised by
high ambient temperature and relative humidity [5]. Corresponding author: Mohammed El Khasmi, Ph.D.,
professor, research field: hormones and metabolism in These conditions of stress, heat and moisture of air dromedary camel. E-mail: [email protected].
14 Effects of Transportation Stress during the Hot-Dry Season on Some Haematological and Physiological Parameters in Moroccan Dromedary Camels (Camelus dromedarius)
acting concurrently on transported animals impair temperature. Animals were captured, loaded and normal body functions, leading to increased morbidity 2 placed in a truck with stocking density about 1/m per
and mortality, poor meat quality and decreased animal. Road trip was made at a speed of 50-60 km/h. productivity [6, 7]. These environmental factors are
All animals were clinically healthy, feed deprived able to provoke excessive generation of ROS (reactive
overnight and were transported to the Tit-Mellil oxygen species) or free radicals as a result of
Municipality slaughterhouse.
increased metabolism [8]. ROS induce oxidative Before loading and transport at 11 h damage of macromolecules, cells and tissues [9] that
(pre-transportation) and immediately after transport at consequently leads to substantial economic losses [10]
13 h (post-transportation), Tr, HR, RR and blood and haematological changes [11].
samples were taken. Two blood samples were
In addition, the mammalian erythrocyte is collected by jugular venipuncture from each camel. enucleated, has a short life span and is very sensitive
One sample was collected in an EDTA-K2 (ethylene to oxidative injury, so, it is considered as an ideal
diamine tetra acetic acid-dipotassium) Vacutainer tube cellular model for study stress damages [12]. Changes
for the determination of Glu, Ht and BCC, whereas in its membrane lipids can affect the erythrocyte shape
the other sample was collected in a heparinized tube by disrupting the balance in area between the two lipid
for the realization of EOF test and the determination leaflets. Furthermore, in several studies, the EOF
of Ca, Pi, Mg and Cor. Following collection, the tubes (erythrocytes osmotic fragility) test was frequently
were gently inverted to ensure mixing of the sample. used to determine the extent of red blood cell
After analysis of Glu, Ht and BCC, and EOF test on haemolysis produced by osmotic stress [13, 14]. The
blood samples, the plasma was separated by extent of the haemolysis is dependent on cell volume,
centrifugation at 750× g for 15 min at 4 °C, pipetted surface area, and functional integrity of cell into aliquots and then stored at -20 °C until analysis of
membranes.
Ca, Pi, Mg and Cor.
To our best knowledge, there are no reports evaluating the EOF and the plasma levels of
2.2 Haematological Parameters
magnesium in camels subjected to transportation
2.2.1 Haematocrit
stress. Therefore, this study was undertaken to The Ht was determined by centrifuging a precise investigate the effects of road transportation under
amount of blood in calibrated haematocrit tubes heat on some haematological [Ht (haematocrit), blood
(Hettich Haematokrit D-7200), the report cell mass/ cells count and EOF (erythrocytes osmotic fragility)]
plasma was expressed as % by direct reading on the and physiological [Tr (rectal temperature), HR (heart)
tube: H (%) = (level of pellet)/(overall height) × 100. and RR (respiratory rates), circulating levels of Cor
2.2.2 Blood Cells Count
(cortisol), Glu (glucose) and minerals] parameters in
A Malassez cell (1 mm 3 ) was used for counting the Moroccan dromedary camels.
blood cells. It comprises five horizontal bands of five
2. Materials and Methods