Journal of Life Sciences Volume 7 Number (6)

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Journal of Life Sciences

Volume 7, Number 1, January 2013 (Serial Number 57)

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J LS

Journal of Life Sciences

Volume 7, Number 1, January 2013 (Serial Number 57)

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

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51 Removal of Different Dyes by Pseudomonas fluorescens

Sewgil Saaduldeen Anwer and Sawan Merkhan

57 Preparation of Organic Selenocystine UsingLocally IsolatedBread 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

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Journal of Life Sciences, ISSN 1934-7391, USA

The Overexpressed FAK (Focal Adhesion Kinase) in

Higher Grade Human Urothelial Tumors

Baz Ahsene1, Ousmaal Mohamed El Fadel1, Mammeri Saâdia2, Zineddine-Charef Amir3, Frederic Boudard4, Frederic Hollande4, Belal Tahar5 and Jean Giaimis5

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



Tumors of the urinary tract are frequent and very often malignant. Although, preservation of the organ could be achieved in certain situations, cystectomy is nevertheless regarded as the standard treatment in the case of bladder cancer [1]. In spite of the progress of the surgical techniques and the introduction of protocols of systemic chemotherapy, many patients with tumors infiltrating the bladder often die within few years from the initial diagnosis. Therefore, the identification of tumor markers and the evaluation of



Corresponding author: Baz Ahsene, Ph.D., professor, research fields: animal physiology and cell signaling. E-mail: [email protected].

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The Overexpressed FAK (Focal Adhesion Kinase) in Higher Grade Human Urothelial Tumors

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implicated in many biological processes, such as proliferation, cell motility and survival as well as in the malignant transformation of normal cells [4, 5]. In cancer cell lines, FAK inhibits apoptosis by activating the PI3-kinase (phosphatidylinositol 3’-OH-kinase)— Akt/PKB (protein kinase B) survival pathway and ultimately by inhibiting the caspase-3 cascade [6, 7]. FAK, PKB and Src are part of a complex network of intracellular signals involved in cancer cell regulation [8]. Conversely, inhibition of FAK expression in different cancer cells makes them loose their attachment and causes apoptosis [9, 10]. In lung tumor cells treated with anti-GD2 ganglioside antibodies, apotosis is elicited by reducing FAK phosphorylation

and activating p38 [11]. Cleavage of FAK by Apo-2L or Fas can induce morphological changes characteristic of apoptotic cells (detachment from the substratum and loss of cell-cell interaction) in Jurkat T and HL60 cells [12]. In conclusion, FAK deregulation might play a role in cancer development and progression [13, 14] and elevated expression of FAK in human tutors has been correlated with increased malignancy and invasiveness [15, 16]. Bladder cancers constitute a major public health preoccupation in Algeria. Men are more affected (79.64%) than women (20.36%) and especially older people as bladder cancers are particularly frequent between 50 and 80 years. The recurrence percentage of urothelial carcinomas is 29.82% in women and 21.52% in men. Since their diagnosis is difficult and often late, new diagnostic and prognostic tools are particularly needed. Therefore, we asked whether changes in FAK expression level in bladder tumors at different stages of dedifferentiation could reflect tumor evolution. To this aim, biopsies of bladder tumors of patients submitted to transurethral resection or cystectomy were recovered immediately after surgical extraction. After staging of the tumors accoroding to the WHO classification [17], FAK expression was evaluated with an anti-FAK specific antibody. Our results show that FAK was overexpressed in bladder cancers as compared to normal bladder tissue.

2. Materials and Methods

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 following informations were recovered: age, weight, gender, tumor antecedents, treatments, tumor description (aspect, size, and number of tumor nodules and presence or not of metastasis). Patients were informed about this research and gave their written agreement.

One half part of each biopsy was used for the histological analysis and the other half part for protein analysis. For histology, tissues were rapidly fixed, dehydrated, embedded in paraffin, and 2 µm to 5 µm serial sections were obtained using a Minot microtome. Sections were then stained with hematoxylin-eosin and observed under a Zeiss Axioskop-40 microscope and microphotographs were taken using an Axiocam digital camera. Determination of the histological type, stage and rank was carried out according to the WHO classification.

2.1 Antibodies

The antibodies used in this study were: mouse anti-FAK monoclonal antibody, clone-77 (1: 1000) (Transduction Laboratories, Lexington, KY, USA), mouse anti-actin monoclonal antibody, clone AC-40 (1: 1000) (Sigma, St Louis, Missouri, USA), and mouse IgG secondary antibody coupled to HRP (Horseradish peroxidase) (1: 5000) (International Chemicon, Inc.).

2.2 Total Protein Extraction

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cocktail, protease inhibitor cocktail (Sigma), vortexed and centrifuged at 4 °C at 14,000 g for 10 min. Supernatants were stored at +4 °C; one aliquot was used for measuring protein concentration according to the Bradford method [18] with a spectrophotometer (UV-1201, Shimadzu) at a wavelength of 595 nm; the rest was kept for western blot analysis.

2.3 SDS-PAGE and Western Blotting

Lysates that were denatured at 100 °C for 4 min and 15 µL (1 µg/µL proteins) were loaded on 8% SDS-PAGE gel [19] and separated at 20 mA/gel to 200 V. Gels were then either stained with Coomassie blue or processed for western blot analysis. For Coomassie blue staining, gels were transferred in 250 mg of Coomassie Shining Blue R250 in 90 mL of 50:50 methanol:distilled water (volume/volume) and 10 mL of acetic acid for 12 h maximum. Gels were then washed in methanol/acetic acid/H2O four times for 4 h maximum. After then, gels were photographed (Fig. 1).

For western blot analysis, separated proteins were transferred onto nitrocellulose transfer membranes (ProteanR BioScience) at 3.5 mA/cm2 intensity with a 15 V constant voltage for 75 min. After protein transfer, membranes were rinsed in PBS-0.1% Tween 20 for 2 min, blocked with PBS-0.1% Tween 20-milk 3% (v/v) for 30 min, and finally rinsed quickly with PBS-0.1% Tween 20 (v/v) for 2 min. Membranes were then incubated with the primary antibodies (anti-FAK or anti-Actin) in PBS-0.1% Tween 20-milk 3% (v/v) overnight at 4 °C, rinsed once with PBS-0.1% Tween 20 (v/v) and then washed once for 15 min and twice for 5 min, and incubated with mouse IgG coupled to HRP at room temperature for 1 h After washes in PBS-0.1% Tween 20 as previously, antibody binding was visualized with ECL (electrogenerated chemiluminescence) detection reagents (Amersham) and bands from autoradiographic films were analyzed with the Photo-Capt MW system and Image J software. FAK expression values were then corrected to Actin

Fig. 1 Protein profiles of normal and tumoral bladder tissues on SDS-PAGE.

150 125 100 75 50 35 25

150 125 100

75 50

25 35

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expression (by dividing the FAK density by the actin correction index) (Fig. 2). The tumor/normal tissue ratio was then calculated by using the Image J Software.

2.4 Statistics

Values of FAK expression were compared using unpaired Fisher’s t-test (significant at P < 0.05; *: P < 0.05; **: P < 0.01; ***: P < 0.001).

3. Results

3.1 Clinical Observations and Histological Classification

Bladder tumors were excised mainly by transurethral endoscopic resection (80% of the patients) or by cystectomy (20%). The following clinical signs were observed (in order of importance): 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 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. 2 Western blot analysis of FAK during urothelial cell retro-differenciation.

massive form; only 3% corresponded to adenocarcinomas. Following the TNM (tumor-node-metastasis) system, tumors were classified as follows: pTa (12%), pT1 (49%), pT2 (28%), pT3 (5%) and pT4 (6%). In situ carcinomas were very rare and accounted for only 1% of all tumors. Moreover, grading according to the tumor cell differentiation showed a clear predominance of grade 2 tumors (Fig. 4); all the tumors excised by cystectomy or cysto-prostatectomy were grade 2 or 3.

The urinary bladder of paired healthy tissues showed a transitional epithelium made of 6-7 layers of regular urothelial cells resting on the basement membrane that separates the epithelium from the connective tissue (lamina propria), the muscularis and adventitia (Fig. 5a). In grade 1 tumors, an increased number of cellular layers in the transitional epithelium were found. Tumor cells were characterized

Fig. 3 Frequency of bladder tumors in all patients (n = 280).

Fig. 4 Tumor localization in the bladder.

Percentages (%

)

Percentages (%

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by anisocytosis, anisocaryosis and rare mitosis; cell polarity was still preserved (Fig. 5b). In grade 2 tumors, the transitional epithelium was very thick, with nuclear anomalies; cells were bulkier with increased mitotic activity and moderate disturbances of cell polarity, which gave to the epithelium a somewhat disorganized aspect (Fig. 5c). In grade 3 tumors, signs of dedifferentiation were obvious, with cytonuclear atypia and architectural disorganization of 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 be detected, its expression increased concomitantly with the grade of the tumor (Figs. 2 and 6).

Specifically, a weak increase in FAK expression was observed in tumors of grade 1 and 2 (1.65; 2.99) (significant for grade 2) as compared to healthy tissues; it became particularly important in grade 3 tumors [16, 18]. In addition, the total protein profiles of samples from healthy tissues and grade 1, 2 and 3 tumors revealed increased staining of a 125 KDa m.w. band which could correspond, beside other proteins, to the FAK protein.

Fig. 6 Quantification of FAK expression in urothelial tumors.*: P < 0.05; **: P < 0.01; ***: P < 0.001 and no 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 ×).

(a) (b)

(c) (d)

E

M

TC E

LB AC

VS I

LB C

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6

4. Discussion

The incidence of bladder cancer is increasing in the industrialized countries and particularly in Western Europe [20]. Currently, the prognosis of bladder tumors is based on anatomo-clinical criteria; however, molecular and genetic analysis could allow an identification of markers whose expression is related to the tumor stage and grade. Such markers could help to identify patients at risk and to predict their response to different therapeutic protocols.

The aim of this work was to assess whether changes in FAK expression could play a potential role in the development and progression of bladder tumors in vivo. Indeed, we show that increased FAK expression is associated with tumors of increasing malignancy and with higher invasive potential. Proteins of the cellular junctions are involved in the maintenance of the architecture and the polarity of epithelial cells [21]. Loss of adhesion due to deregulation of factors localized at focal adhesion sites, such as FAK, can facilitate migration of tumor cells and therefore formation of metastasis [22]. Indeed, increased phosphorylation of FAK favors metastatic invasion of colon cancer cells [23, 24]. Overexpression and/or activation of Src and FAK increase cellular proliferation, survival and metastasis [25, 26]. FAK is involved in complex regulatory pathways, implying adhesion with the extracellular matrix, but also growth factors, hormones and chemokines [27]. For instance, interactions between FAK and Src or Bcr/Abl have been described [13]; in Src-induced colon cancer cells EPS8, FAK expression was deregulated through mTOR and STAT3, both at the level of gene transcription and translation [28].

In the data, the weak expression of FAK in grade 1 and 2 tumors and its increase in grade 3 tumors suggests that cell migration, loss of urothelial tissue integrity and disorganization could be due at least in part to FAK activity. Indeed, in conditional KO mice, loss of Fak upon treatment with 4-hydroxy-

tamoxifene suppressed chemically-induced tumor formation. Moreover, loss of Fak inhibited malignant progression of already formed benign tumors; loss of Fak was also associated to a reduction of in vitro migration and with keratinocytes apoptosis both in vitro and in vivo [29]. Furthermore, high levels of FAK were correlated with migration of cancer cells, invasion, and ability to metastasize [30].

The authors’ study, by associating clinical, histological and biochemical data, highlights the implication of FAK in the development of malignant features in human bladder carcinomas in vivo. These results are in agreement with previous work carried out in other type of cancers [8, 23, 30-32].

5. Conclusion

The development of new tools targeting critical molecular anomalies involved in tumor progression is very much needed. Indeed, the rational use of tumor markers may allow the choice of individualized therapeutic strategies for each patient affected by bladder cancer. However, currently, many markers are associated with tumors with different staging and grading; to be really useful, these markers must be validated by multicentric prospective studies.

Acknowledgments

The authors would like to thank Prof. Jacques Mercier (Montpellier, France) and Prof. Jean Pierre Bali (Montpellier, France) for helpful comments. This project was supported by DAHR (the Development Agency of Health Research), Oran (Algeria) and by the Ministry of Research of Algeria.

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focal adhesion proteins in signal transduction and oncogenesis, Crit. Rev. Oncog. 8 (1997) 343-358.

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[20] B. Lobel, C.C. Abbou, M.A. Brausi, R.C. Flanigan, S. Kameyama, H.I. Scher, et al., Recommendations for the diagnosis, treatment, and follow-up of cancer of the bladder, Prog. Urol. 8 (1998) 590-592.

[21] A. Zahraoui, Tight junctions, a platform regulating cell proliferation and polarity, Med. Sci. 20 (2004) 580-585. [22] D. Ilié, C.H. Damsky, T. Yamamoto, Focal adhesion

kinase: At the crossroads of signal transduction, J. Cell Sci. 110 (1997) 401-407.

[23] L.V. Owens, L. Xu, G.A. Dent, X. Yang, G.C. Sturge, R.J. Craven, et al., Focal adhesion kinase as a marker of invasive potential in differentiated human thyroid cancer, Ann. Surg. Oncol. 3 (1996) 100-105.

[24] M. Agochiya, V.G. Brunton, D.W. Owens, E.K. Parkinson, C. Paraskeva, W.N. Keith, et al., Increased dosage and amplification of the focal adhesion kinase gene in human cancer cells, Oncogene 18 (1999) 5646-5653.

[25] J.T. Parsons, Focal adhesion kinase: The first ten years, J. Cell Sci. 116 (2003) 1409-1416.

[26] S.K. Hanks, L. Ryzhova, N.Y. Shin, J. Brábek, Focal adhesion kinase signaling activities and their implications in the control of cell survival and motility, Front Biosci. 8 (2003) d982-d996.

[27] E. Zamir, B. Geiger, Molecular complexity and dynamics of cell-matrix adhesions, J. Cell Sci. 114 (2001) 3583- 3590.

[28] M.C. Maa, J.C. Lee, Y.J. Chen, S.T. Wang, C.C. Huang, N.H. Chow, et al., Eps8 facilitates cellular growth and motility of colon cancer cells by increasing the expression and activity of focal adhesion kinase, J. Biol. Chem. 282 (2007) 19399-19409.

[29] G.W. McLean, N.H. Komiyama, B. Serrels, H. Asano, L. Reynolds, F. Conti, et al., Specific deletion of focal adhesion kinase suppresses tumor formation and blocks malignant progression, Genes Dev. 18 (2004) 2998-3003.

[30] E. Toton, M. Rybczynska, The characteristics of focal adhesion kinase (FAK) and its role in carcinogenesis, Postepy Hig. Med. Dosw. 61 (2007) 303-309.

[31] M. Agochiya, V.G. Brunton, D.W. Owens, E.K. Parkinson, C. Paraskeva, W.N. Keith, et al., Increased dosage and amplification of the focal adhesion kinase gene in human cancer cells, Oncogene 18 (1999) 5646-5653.

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Jan. 2013, Vol. 7, No. 1, pp. 8-12

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



The main components in the structural organization of DNA in the eukaryotic cells are histones. The special evolutionary conservatism of amino acids sequences of histones enriched by arginine in the eukaryotic kingdom is marked. Extremely reactive guanidine group in δ-position is structural feature of arginine. Guanidine group of arginine possesses the strongest basic properties in comparison with lysine. This group actively is exposed to various sorts of modifications which lead to change of interrelations of supramolecular complexes in process onto- and philogenesis of eukaryotes. As to bacterial cells, E. coli is investigated most full.

It is proved that the histone-like proteins is formed the structure of nucleoid bacteria. Similarity between histone-like proteins of prokaryotes and histone proteins of eukaryotes testifies that packing of DNA at



Corresponding author: Ivanova Evilina, Ph.D., research field: biochemistry. E-mail: [email protected].

eu- and prokaryotic organisms can have the common features. It is assumed that Arg-X proteolysis functions are in a chromatin of eukaryotic cells. Probably the Arg-X proteolysis functions in a bacterial chromosome taking an active part in reorganization structure of nucleoid.

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.

2. Materials and Methods

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of Arginine in Life Cycle in Population of E. coli

supramolecular structures were fractionated from bacterial cells according to the method [4]. Proteome structures from E. coli were fractionated by breaking of the weak and strong bonds of supramolecular structures. It was done by increasing ionic strength of solution: 0.14 M NaCl; 0.35 M NaCl; 2 M NaCl; 6 M

guanidine-hydrochloride with 0.004% β-mercaptoethanol at 0.01 M Tris-HCl buffer (pH 6.8). Extraction of proteins by increasing the ionic strength of the salt gradient, leading to a weakening of the electrostatic interaction between proteins and adsorbent, are the usual methods of protein chemistry [5]. The fraction of Cp (cytoplasm) was isolated by 0.14 M NaCl. The fraction of supramolecular structure of loosely bound with cellular rest (Sp I) was received by the extraction of 0.35 M NaCl. The fraction of supramolecular structure of tightly bound with cellular rest (Sp II) was isolated by 2 М NaCl. CR (Cellular

rest) was obtained by extraction 6 МGu·HCL

(guanidine hydrochloride) and 0.004% β-mercaptoethanol [4, 6]. The amount of protein in the supramolecular structures was determined by Bradford in our modification [4]. Arg-X activity was valuated from digestion of Arg-X bonds in the protamine Salmine-AI (Merk, Darmstadt, Germany), a protein enriched by Arg. This protein comprises 33

amino acids: 22-Arg, 4 Ser, 3 Pro, 2 Gly and 2 Val. Activity Arg-X proteolysis was expressed in nM of arginine/(μ protein·s). The numbers and points on the graphs represent the arithmetic mean data.

3. Results and Discussion

3.1 Physiology-Biochemical State of E. coli Cells

The understanding of biological processes is reached much more easily by means of modeling organisms. In a bacterial cell, the chromosome is packed in the form of the compact structure connected with a membrane. This DNA-membrane complex provides structural packing of a chromosome, replication and segregation [7].

In an active growth phase (the period from 50 min to 190 min) cells of bacteria grow with the highest speed in our experiment (Fig. 1). At gradual depletion of necessary nutrients and accumulation of products of metabolism, the growth rate of bacteria is decreasing (from 190 min to 330 min—delay phase). Then growth of bacteria is stopping (from 330 min to 430 min), the culture is passing into a stationary phase. It is believed that when the culture of bacteria pass to a stationary phase the differentiation program is started, leading to that cells become metabolic less active and

Fig. 1 Dynamics of increasing the population density of cells E. coli in the life cycle. Active

Amount of cells in 1.5

mL

eluat

e

Stationary

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Features of Structurization at Participation of Guanidine Groups of Arginine in Life Cycle in Population of E. coli

10

more resistant to stressful factors [8]. Many functions are induced upon transition of culture to the stationary phase. And it’s activated at the limitation of nutrients [8]. Under these conditions, the expression of most bacterial genes is significantly reduced. However, there is an induction of an expression of a large number of other genes and synthesis of specific proteins, first of all what provide stability of bacteria to various adverse conditions is stimulated [8]. Thus life cycle of bacteria includes the periods of the active growth alternating with the periods of delay and the termination of growth (Fig. 1). In natural conditions, bacteria are seldom lived in abundance conditions, allowing exponential growth [8]. Short periods of rapid growth are alternating with long periods of starvation (stationary phase); the cells are undergoing various unfavorable factors for their life activity. Inthese conditions the bacteria should be lived for a long time. And then come back to an exponential phase when influence of starvation and other adverse effects will be removed [8].

This period also is characterized by activation of molecular mechanisms of adaptation of microorganisms to a stress. The fact that cells in the stationary phase are essentially steadier against

stressful influences testifies that in the specified conditions in cells there is a raised expression of the genes necessary for protection [8]. In the conditions of a stress microorganisms become hardier and, their pathogenic factor is increasing [9].

3.2 Arg-X Protease-Sensitive in Supramolecular Structures of E. coli Cells

Studying of thin molecular and supramolecular structures is capable to deepen our understanding of the spatial-temporary and functional organization of a prokaryotic cell. As a matter of fact, cellular level of the organization of life activity of a bacterium can be considered from a position of well organized “associate” of molecules, the supramolecular structures which are constantly cooperating with each other and environment. In Albrecht-Buhler’s opinion [10], supramolecular descriptions of form-operating processes are valuable as interactions of many molecules are integrated into them because signals along their linear structures at the expense of association and a dissociation of molecules can move. We discuss results of experiment in Fig. 2 from these positions. Fig. 2 shows the characteristics of spatial-temporal Arg-X protease—sensitivity of.

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).

Active Delay Stationary

Growth Phases

nM Ar

g./(s

·

μ

protein) on ce

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of Arginine in Life Cycle in Population of E. coli

supramolecular structures of bacterial cells of E. coli. The data was shown that in the period of transition to stationary growth phase (phase of active stress) in the cytoplasm was increased cyclic splash of Arg-X proteolysis. A number of studies have shown that in the stationary growth phase the qualitative and quantitative changes in protein composition of nucleoid influence on DNA compaction [8]. At delay and termination of growth, the cell culture of E. coli undergoes significant morphological changes. Cell shape is changing: if during the period of rapid growth of the culture, they have a rod-shaped form, at the delay of growth of the culture, they become much smaller and almost spherical. It is a consequence of several cell divisions without an increase incellmass It is believed that the sharp decrease in cell size can contribute to the survival of bacteria by increasing their number. The cytoplasm of cells is condensing; the volume of the periplasm is increasing [8]. The rate of proteolysis of the cellular proteins is also increasing by at least several times during starvation of cells in the stationary phase of growth. The amino acids released as a result proteolysis of proteins are used for synthesis of new proteins [8]. It is quite possible that during of this period biogenic amines are forming. For example, from arginine are forming agmatine. Agmatine in a certain dose is possessing poisonous properties. Global changes in gene expression at transition of cells to a stationary growth phase occur at each stage of an expression of genes and include changes in conformation of nucleoid, the apparatus of transcription and translation. Chromosomal DNA of E. coli is associated with 10 main types of structural proteins, forming nucleoid. These proteins are now often referred to as nucleoid proteins; they play the important role in the regulation of such processes as replication, recombination and transcription which are required for cells [8]. At transition from the culture to a stationary phase, topological changes are occurring in chromosome of the starving cells, correlating with reduction of the general level of an expression of

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Features of Structurization at Participation of Guanidine Groups of Arginine in Life Cycle in Population of E. coli

12

According to literature, it is known that at long action of adverse factors, the increase in activity of arginine-decarboxylase is observed. As a result of decarboxylation reaction free arginine turns in biogenic amine-agmatin. In the conditions of the stationary phase of life cycle of bacteria, the induction of expression of the genes providing synthesis of proteins which are necessary for their stability to adverse conditions is being carried out. During this period, there is a high continuous activity of the Arg-X processing at the level of “cytoskeleton” of the cell and bright cyclic activity in the cytoplasm is noted.

4. Conclusion

It is known that in the conditions of a stationary phase of life cycle of bacteria, an induction of expression of the genes providing synthesis of proteins which are necessary for their stability to adverse conditions is being carried out. During this period high continuous activity of Arg-X of processing at level of “cytoskeleton” of the cell is noted. The knowledge of mechanisms of regulation of expression of genes will allow finding ways of target modification of guanidine groups in different growth phases of cells necessary for nanotechnological designs.

References

[1] D.B. Myrphy, J.T. Pembroke, Transfer of the IncJ plasmid R391 to recombination deficient Escherichia coli K12: Evidence that R391 behaves as a conjugal transposon, FEMS Microbiology Letters 134 (1995) 153-158.

[2] T. Maniatis, E.F. Fritsch, J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1982. [3] E.A. Ivanova, G.H. Vafina, Method of selection of plant

cell nuclei, RF Patent, 1701747 B48 (1991).

[4] E.A. Ivanova, G.H. Vafina, A method of producing nuclear fractions with proteinase and inhibitory activity, RF Patent, 1733471 B18 (1992).

[5] R.K. Scopes, Protein Purification: Principle and Practice, Springer-Verlag, New York, NY, 1982.

[6] E.A. Ivanova, G.H. Vafina, T.S. Tropynina, A method of producing fractions of E. coli cell with proteinase activity, RF Patent, 2410428 B3 (2011).

[7] S. Hiraga, T. Ogura, H. Niki, C. Ichinose, H. Mori, Positioning of replicated chromosomes in Escherichia coli, Journal of Bacteriology 172 (1990) 31-39.

[8] I.A. Khmel, Regulation of expression of bacterial genes in the absence of active cell growth, Russian Journal of Genetics 41 (9) (2005) 968-984.

[9] A.G. Tkachenko, Bacteria and stress, Science of the Ural 5 (2010) 4-5.

[10] G. Albrecht-Buhler, In defense of “nonmolecular” cell biology, International Review of Cytology 120 (1990) 191-241.

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Effects of Transportation Stress during the Hot-Dry

Season on Some Haematological and Physiological

Parameters in Moroccan Dromedary Camels (

Camelus

dromedarius

)

Mohammed El Khasmi1, Youssef Chakir1, Fouad Riad1, Abdallah Safwate1, El Hassane Tahri1, Mohamed Farh1, Najia El Abbadi2, Rachid Abouhafs3 and Bernard Faye4

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



Road transportation of animals is a potent stressor, which results from vehicle motion, noise and vibration, and can change several of the animal’s physiological systems (e.g. cardiovascular, immune and endocrine via the activation of HPAA (Hypothalamo-

Corresponding author: Mohammed El Khasmi, Ph.D.,

professor, research field: hormones and metabolism in dromedary camel. E-mail: [email protected].

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Effects of Transportation Stress during the Hot-Dry Season on Some Haematological and Physiological Parameters in Moroccan Dromedary Camels (Camelus dromedarius) 14

acting concurrently on transported animals impair normal body functions, leading to increased morbidity and mortality, poor meat quality and decreased productivity [6, 7]. These environmental factors are able to provoke excessive generation of ROS (reactive oxygen species) or free radicals as a result of increased metabolism [8]. ROS induce oxidative damage of macromolecules, cells and tissues [9] that consequently leads to substantial economic losses [10] and haematological changes [11].

In addition, the mammalian erythrocyte is enucleated, has a short life span and is very sensitive to oxidative injury, so, it is considered as an ideal cellular model for study stress damages [12]. Changes in its membrane lipids can affect the erythrocyte shape by disrupting the balance in area between the two lipid leaflets. Furthermore, in several studies, the EOF (erythrocytes osmotic fragility) test was frequently used to determine the extent of red blood cell haemolysis produced by osmotic stress [13, 14]. The extent of the haemolysis is dependent on cell volume, surface area, and functional integrity of cell membranes.

To our best knowledge, there are no reports evaluating the EOF and the plasma levels of magnesium in camels subjected to transportation stress. Therefore, this study was undertaken to investigate the effects of road transportation under heat on some haematological [Ht (haematocrit), blood cells count and EOF (erythrocytes osmotic fragility)] and physiological [Tr (rectal temperature), HR (heart) and RR (respiratory rates), circulating levels of Cor (cortisol), Glu (glucose) and minerals] parameters in Moroccan dromedary camels.

2. Materials and Methods

2.1 Animals, Transportation and Blood Sampling

To assess some haematological and physiological stress responses, 17 male camels (6 to 7 years of age, average weight of 430 kg ± 40 kg) were subjected to 2 h stressful road transportation at a high ambient

temperature. Animals were captured, loaded and placed in a truck with stocking density about 1/m2 per animal. Road trip was made at a speed of 50-60 km/h. All animals were clinically healthy, feed deprived overnight and were transported to the Tit-Mellil Municipality slaughterhouse.

Before loading and transport at 11 h (pre-transportation) and immediately after transport at 13 h (post-transportation), Tr, HR, RR and blood samples were taken. Two blood samples were collected by jugular venipuncture from each camel. One sample was collected in an EDTA-K2 (ethylene diamine tetra acetic acid-dipotassium) Vacutainer tube for the determination of Glu, Ht and BCC, whereas the other sample was collected in a heparinized tube for the realization of EOF test and the determination of Ca, Pi, Mg and Cor. Following collection, the tubes were gently inverted to ensure mixing of the sample. After analysis of Glu, Ht and BCC, and EOF test on blood samples, the plasma was separated by centrifugation at 750× g for 15 min at 4 °C, pipetted into aliquots and then stored at -20 °C until analysis of Ca, Pi, Mg and Cor.

2.2 Haematological Parameters

2.2.1 Haematocrit

The Ht was determined by centrifuging a precise amount of blood in calibrated haematocrit tubes (Hettich Haematokrit D-7200), the report cell mass/ plasma was expressed as % by direct reading on the tube: H (%) = (level of pellet)/(overall height) × 100.

2.2.2 Blood Cells Count

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Physiological Parameters in Moroccan Dromedary Camels (Camelus dromedarius)

As the volume of a rectangle is 1/100 mm3 and the dilution is 1/200, the final result is therefore: m × 100 × 200 erythrocytes/mm3 or m × 100 × 200 × 103 erythrocytes/mL of blood. Leukocytes were counted in five horizontal bands (= N’). As the volume of a horizontal band is 1/10 mm3, for five counted bands the volume becomes 1/2 mm3. There are therefore N’ × 2 leucocytes/mm3 of blood diluted to 1/20. The end result is therefore: N’ × 2 × 20 leucocytes/mm3 or N’ × 2 × 20 × 103 leucocytes/mL of blood.

2.2.3 Erythrocyte Osmotic Fragility

The osmotic fragility of erythrocytes was determined by the procedure described by Oyewale [15] and Oladele et al. [16]. Briefly, sodium chloride stock solution (pH 7.4) was prepared in volumes of 500 mL for each of the sample in concentration, ranging from 0.1% to 0.9%. A set of 10 test tubes was used and each tube contained 5 mL of the corresponding NaCl (sodium chloride) concentration from the stock solution. The test tubes were then labelled with corresponding concentrations and arranged serially in a rack of 10 tubes. 1 ml pipette was then used to transfer exactly 0.02 mL of each blood sample into each of the 10 test tubes in a set. The contents of the test tubes were gently mixed by inverting the test tubes five times and allowing them to stand at room temperature for 30 min. Thereafter, the contents of the test tubes were centrifuged at 1500× g for 15 min. The supernatant was then transferred into a glass cup and measure data wave length of 540 nm using a spectrophotometer by reading the absorbance. The percent haemolysis was calculated according to Fraukner and King [17] as follows:

Percent haemolysis = [Optical density of test/ Optical density of standard (distilled water)] × 100.

EOF curve was obtained by plotting percent haemolysis against the saline concentrations.

2.3 Physiological Parameters

2.3.1 Rectal Temperature and Heart and Respiratory Rates

Tr was taken in the rectum with a thermometer. The

HR (beats/min) was determined by auscultation of the heart area or pulse by feeling the tibial, femoral and coccygeal arteries. The RR (breaths/min) was determined by inspection and auscultation of the respiratory movements of the trachea.

2.3.2 Dosage of Glucose, Minerals and Cortisol Blood concentrations of Glu and plasma levels of Ca, Pi and Mg (magnesium) were measured using commercial kits. The plasma Cor concentrations were analyzed by RIA (radioimmunoassay) in medical and biological application laboratory (National Center of Energy of Nuclear Science and Technology in Maamoura, Morocco) by using commercially available coated RIA tubes for human Cor. These human kits have been used in previous experiments in dromedary camels [2], and were purchased from DIAsource (Immunoassays S.A., Nivelles, Belgium). Validation for hormone assays included limits of detection, and precision in standard curve following sample dilution, between and within-assays.

2.4 Statistical Analysis

All data obtained were subjected to statistical analysis using Student’s t-test. Data were expressed as mean ± standard error of mean. Values of P < 0.05 were considered significant.

3. Results

3.1 Meteorological Conditions

The ambient temperature registered in the study area during our experimentation ranged between 31.7 °C ± 2.5 °C and 34.2 °C ± 2.1 °C, while the relative humidity fluctuated between 55.4% ± 6.0% and 64.3% ± 4.9%.

3.2 Haematological Parameters

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vs. 34.6 ± 2.5 and 11.51 ± 0.98 vs. 8.4 ± 0.85, respectively), without any significant change of white blood cells count (× 1 million/mL) (6.25 ± 1.82 vs. 8.46 ± 2.14, respectively) (Fig. 1).

The curve of osmotic fragility of camel’s erythrocytes is shown in Fig. 2. The pattern of the erythrocytes response to haemolysis was basically increased with decreasing concentration of saline solution. The test of haemolysis in various hypo-osmotic solutions of NaCl showed that

erythrocytes of our camels subjected to transportation stress were characterized by a signiﬁcant shift of the curves to the right, indicating a decreased osmotic resistance of erythrocytes by comparison with that measured before transportation (Fig. 2). Thus, for example, hemolysis started at 0.4% and 0.6% NaCl before and after transportation respectively. In both unstressed (pre-transportation) and stressed (post-transportation) camels, 0.1% NaCl resulted in 100% haemolysis (Fig. 2).

Erythrocytes (x1milliard/mL) Leucocytes (x1million/mL) Hematocrit (%)

He

Fig. 1 Blood cells count and haematocrit in dromedary camels before transport (pre-transportation) and immediately after transport (post-transportation). (Each point represents the mean ± SEM. *: P < 0.05: differences between pre-transportation and post-transportation values).

Fig. 2 The erythrocyte osmotic fragility in dromedary camels before transport (pre-transportation) and immediately after transport (post-transportation) at a pH value of 7.4 and temperature of 37 °C. (Each point represents the mean ± SEM. *: P

< 0.05: differences between pre-transportation and post-transportation values).

(billion/m) (billion/m)

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3.3 Physiological Parameters

Stress transportation caused a significant increase (P < 0.05) of plasma levels of Tr (°C), HR (beats/min) and RR (breaths/min) comparatively to values observed before stress conditions (40.2 ± 0.4 vs. 38.1 ± 0.3, 58 ± 4 vs. 46 ± 3 and 17 ± 2 vs. 11 ± 2, respectively) (Fig. 3).

Under stress transportation, the dromedary camels

showed a significant increase (P < 0.05) of glycaemia (mM) (Fig. 4) and Cor (ng/mL) (Fig. 5) (9.7 ± 1.2 vs. 6.4 ± 1.1 and 220 ± 30 vs. 137 ± 20, respectively) and a significant decrease (P < 0.05) of magnesia (mM) (Fig. 4) (0.5 ± 0.1 vs. 0.9 ± 0.1) by comparison with values measured in the same animals before transportation, without any significant change of plasma levels of Ca and Pi (2.4 ± 0.2 vs. 2.7 ± 0.2 and 1.1 ± 0.2 vs. 1.5 ± 0.3, respectively) (Fig. 4).

0 10 20 30 40 50 60 70

Rectal temperature (°C) Respiratory rate (breaths/min) Heart rate (beats/min)

P

h

y

sio

lo

g

ica

l p

a

ra

m

eter v

a

lu

e

Pre-transportation

Post-transportation

٭

٭ ٭

Fig. 3 Rectal temperature, respiratory rate and heart rate in dromedary camels before transport (pre-transportation) and immediately after transport (post-transportation). (Each point represents the mean ± SEM. *: P < 0.05: differences between pre-transportation and post-transportation values).

0 2 4 6 8 10 12

Calcium Phosphorus Magnesium Glucose

C

ir

cul

at

in

g l

ev

el

s (

m

M

)

Pre-transportation Post-transportation

٭

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4. Discussion

4.1 Physiological Parameters and Blood Cells Count

The authors’ results obtained before loading and transportation (Fig. 1) were similar and within the normal ranges of values recorded in camels for the BCC, Ht [18-20] and EOF [18, 21], which suggested that our camels were healthy.

The significant increase of Tr, HR, Ht and RBC number in response to transportation in camels (Fig. 3) has been documented in numerous species such as calf [22-24], horse [25], bull [26], goat [14, 27-29] and sheep [30, 31], and has been used to evaluate the stress [32]. The hyperthermia during transport under heat may induce a water loss caused by thermo regulation and urination, then contribute to dehydration and increase in RBC and Ht. This increase may be due to a splenic contraction rather than to dehydration [33]. In fact, acute exposure of animals to stressful stimulation is attended by a significant activation of the sympathetic-adrenal medullary system, including increased synthesis, circulating levels and release of catecholamines into the circulation [34], resulting in splenic contraction and the release of RBC into the circulation. This mechanism is induced by the action of catecholamines on α-adrenergic receptors located in the splenic capsule [27, 28]. However, in male cynomolgus monkeys, after transport in cages, hematological parameters of white blood cell count, RBC count, hemoglobin concentration, and Ht values were within the limits of reference range on arrival [35].

4.2 Plasma Glucose

The increase of plasma Glu levels under road transportation and heat observed in our camels (Fig. 4) agrees with the results found in horse [25], goat [14, 27-29] and Holstein calf [24]. This hyperglycaemia may be primarily due to an activation of the sympathetic nervous system, catecholamines secretion [36] which is able to decrease the glycogen reserves by stimulating the glycogenolysis [37, 38]. However,

a depletion of muscle glycogen could influence the post-mortem metabolic changes then the quality of meat [2, 39-41]. On the other hand, the release of Cor a neoglucogenetic hormone caused by stress may contribute to hyperglycaemia. Thus, in goats, the elevation of plasma Glu is preceded by an increase in the plasma Cor concentrations [42] and remained higher for approximately 3 h after 2 h transportation [43].

4.3 Circulating Cortisol

The increase of plasma Cor as adrenocortical response by a road transportation and heat observed in our camels (Fig. 5) agrees with that noted in the same species [2, 3, 44] and those reported under the same stress conditions in several other species. Thus, in sheep [45, 46], pig [47, 48], horse [25], goat [14, 27-29, 43], bull [26], calf [22, 24] and monkey [35] Cor levels were higher in blood samples collected after transport than in samples collected before transport. In Japanese Black steer calves, the plasma Cor level increased immediately during transport around the mountainous area and was significantly higher (50.8 ng/mL ± 6.7 ng/mL) at the end of transport compared with the control calves (14.7 ng/mL ± 6.7 ng/mL) [49]. However, road transportation did not induce any increase of circulating Cor, in pig [49, 50]. In addition, in the sheep, apart from changes in hydration (decrease of body weight and increase of Ht), plasma levels of Cor after 12, 30, or 48 h of transport did not show any significant increase [51].

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0 50 100 150 200 250 300

Pre-transportation Post-transportation

P

la

sm

a

C

o

rt

iso

l le

v

els

(

n

g

/m

l)

٭

Fig. 5 Plasma levels of cortisol in dromedary camels before transport (pre-transportation) and immediately after transport (post-transportation). (Each point represents the mean ± SEM. *: P < 0.05: differences between pre-transportation and post-transportation values).

fragment (10 mg/kg) over 1 h induced high plasma levels of cortisol since 30th min of infusion until 60 min after the cessation of the infusion. During stress, the hypothalamus receives direct stimulation of the limbic system and stimulation from the noradrenergic locus coeruleus and nucleus of the solitary tract. In response to these stimuli, the hypothalamus releases the corticotropin-releasing hormone in the hypothamo-pituitary portal system for activate the adenohypophysis. This gland responds by secreting ACTH which stimulates the synthesis and the release of glucocorticoïds by adrenal glands [53].

It is largely documented that Cor responses to stress and the reaction of animals to stressors may be influenced by the sex [54], the amount of visceral adipose tissue [55] and possibly by other factors such splanchnic innervation of the adrenal gland [56], lighting, food intake [57] and age [44]. The impact of stress can be influenced by the duration and the intensity of transport. So, according to Alberghina et al. [58], the degree of short transport stress was greater in young horses than in older ones, however young horses were shown to be better adapted than

old horses to long transport.

4.4 Erythrocytes Osmotic Fragility

In the present study: the curve of unstressed camels (pre-transportation) started haemolysis at 0.4% NaCl (Fig. 2), which demonstrates that camels have more resistant erythrocytes than that of sheep, cattle and humans which commence haemolysis at 0.85%,

0.70% and 0.55% NaCl, respectively [59]. On the other hand, it has been established that erythrocytes of males are more susceptible to haemolysis than those of females in domestic fowl [60, 61], cattle [62] and camel [21]. According to Amin et al. [18], in camel, the osmotic resistance increased significantly during the green season.

It is well documented that the erythrocytes of camels are resistant to haemolysis when compared with sheep, goat, man and other animals [63-65]. Camel erythrocytes are able to expand to 240% of their original volume without rupturing in hypotonic solutions [66] and have an oval shape [67]. So, this

animal has an exceptional ability to rapidly replace

(ng

/mL

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water lost during prolonged periods of dehydration within a few minutes of access to drinking water without haemolysis [68]. The exceptional osmotic resistance of camel erythrocytes may be due to the high concentration of total lipids, cholesterol, proteins, sphingomyelin and phosphatidylcholine in the erythrocyte membranes of camels when compared

with the concentrations of these parameters in the erythrocyte membranes of sheep and goats [69]. In addition, the augmented water-binding associated with the high hydrophilicity of camel haemoglobin may contribute to this high osmotic resistance. In fact, according to Bogner et al. [70], the proportion of osmotically non-removable water in camel erythrocytes is nearly 3-fold greater than that in human erythrocytes (approximately 65 vs. approximately 20%). On the other hand, erythrocytes of camels show a very low water contents (1.1-1.3 g water/g dry mass) [71-74] and a difference in the major intrinsic membrane water-soluble protein

“spectrin” which appears to be very tightly bound to the membrane by comparison to those of humans and bovine species [75].

The increase of EOF by road transportation stress under heat observed in the present study (Fig. 2) agrees with the results observed in the same conditions in horse [25], goat [14, 27-29], calf [24] and pig [13]. Transportation and heat stress can exert both “physical”, “physiological” and “psychological” stressful, then increase EOF by causing erythrocytes destruction. In fact, erythrocytes are very sensitive to oxidative injury [12] and an increase in free-radical generation as a result of stress depletes antioxidant on the erythrocyte membrane; thus, causes oxidative damage of the membrane proteins and lipids then induces haemolysis [76]. The transport and/or heat stress conditions can induce the metabolic changes that are involved in the induction of oxidative stress by enhancing the formation of ROS (reactive oxygen species). In dromedary camels, road transportation under hot ambient temperature might cause an

oxidative challenge by inducting a significant increase of plasma concentrations of malondialdehyde and whole blood GSH-Px (glutathione peroxidase) activities [77] and increase sceptibility for infections such as pneumonia [78]. In cattle, it has been reported that oxidative stress has been evidenced by excessive accumulation of leukocyte lipid oxidation products under transport [79] and by higher whole blood GSH-Px activities under heat during summer months in cattle [12] indicating an increase of lipid peroxidation levels [80]. Other factors may contribute to the increase in haemolysis and include changes in blood pH and increase of muscles consumption of oxygen under stress [81], resulting a free radical production, then, an erythrocytes destruction [28]. Furthermore, the high plasma levels of circulating Cor in our transported camels may be involved in their elevated hemolysis by alteration of erythrocyte membranes. In fact, in rat, three-fold cortisol injection (25 mg/kg, once per day) potentiated stress-induced changes in the processes of free radical oxidation of lipids in the hypothalamus [82]. In addition, it had been reported in mammals that administration of glucocorticoid hormones caused lipid peroxidation by decreasing the nonenzymatic antioxidant capacity and suppressing the enzymatic antioxidant systems in the liver [83], erythrocytes [84], skeletal muscle and lymphoid organs [85].

4.5 Magnesium and Oxidant Stress

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compromised in hypomagnesemia and oxidative tissue destruction results. In mice, hypomagnesemia lowers the activity of superoxide dismutase and catalase, glutathione S-transferase and glutathione reductase activities in red cells [88]. In addition, Mg showed favourable effects on haematological and other biochemical parameters under oxidative stress induced by arsenic poisoning in humans [89]. However, in pig, transportation stress did not induce any significant variation of plasma Mg levels [90]. Finally, causes of decreased magnesium may include excessive

urinary losses, renal dysfunction, hypercalcemia or hypophosphatemia, hyperthyroidism, hyperaldosteronism, diuresis, decreased intake of Mg, increased intestinal losses, and pancreatitis [91].

On the other hand, heat and transportation stress has been reported to impair absorption of AA (ascorbic acid) and Mg [92]: thus, lipid peroxidation increases in the plasma and tissues leading to damage of cell membranes [93]. AA administration in pigs subjected to road transportation during the harmatan wind and goats during the hot-dry season in the Northern Guinea Savannah zone of Nigeria has been shown to decrease hemolysis and reduced oxidative damage to erythrocytes [13, 14]. This finding supports the earlier reports by Adenkola and Ayo [13] in pigs transported at ambient temperature of 19.33 °C ± 2.9 °C and Minka and Ayo [14] in goats transported at ambient temperatures between 28.2 °C and 40.1 °C, that the administration of AA prior to road transportation ameliorated the effects of the stress due to the journey on EOF, by a marked reduction in erythrocyte haemolysis. In addition, Tauler et al. [28] and Kraus et al. [94] demonstrated that AA was capable of stabilizing membrane integrity of cells and that it decreases membrane susceptibility to lipid peroxidation encountered during stress. AA also has been demonstrated to be capable to inhibit the synthesis of Cor [95], detoxify free radicals by donating free hydrogen ions [96], reduce the capacity of oxygen consumption by stressed cells: decrease

heat load and increase heat loss encountered during stress [97].

5. Conclusion

Transportation under heat may cause significant physiological (increase of Tr, HR, RR and circulating Cor and Glu levels, decrease of circulating Mg levels) and haematological (increase of RBC number, Ht and EOF) changes in camels. For the first time, the potential use of EOF as a diagnostic tool in road transportation stress has been demonstrated in camels in the present investigation. Further studies of an eventual reduction of these stress responses by other parameters such as vitamin C and tocopherols in camel are needed.

Acknowledgments

The authors would like to thank the Governor of Mediouna for access to Tit-Mellil Municipality slaughterhouse and for assisting in samples collection.

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