ISSN (Print): 2328-3947

ISSN (Online): 2328-3955

Editor-in-Chief: Hari K. Koul

Website: http://www.sciepub.com/journal/AJBR

   

Article

Granule Associated DNase in T-Lymphocytes from Patients with Inflammatory Disease

1Department of Biochemistry, Institute of Fundamental Medicine and Biology, Kazan (Volga Region) Federal University, Kazan, Russian Federation

2Laboratoire de Biochimie et de Microbiologie, Ecole Normale Supérieure de Natitingou (ENS), Université de Parakou, Parakou-Bénin

3Clinic of allergic diseases of Kazan Research Institute of Epidemiology and Microbiology, Kazan, Russian Federation


American Journal of Biomedical Research. 2016, 4(4), 87-93
doi: 10.12691/ajbr-4-4-2
Copyright © 2016 Science and Education Publishing

Cite this paper:
Bera C. Скрипов, Cyrille A. VODOUNON, Irina D. RESHETNIKOVA, Boris B. LEGBA, Zinaida I. ABRAMOVA. Granule Associated DNase in T-Lymphocytes from Patients with Inflammatory Disease. American Journal of Biomedical Research. 2016; 4(4):87-93. doi: 10.12691/ajbr-4-4-2.

Correspondence to: Cyrille  A. VODOUNON, Department of Biochemistry, Institute of Fundamental Medicine and Biology, Kazan (Volga Region) Federal University, Kazan, Russian Federation. Email: sweetiebj@yahoo.fr

Abstract

Recent data highlight the undeniable role of programmed cell death type I of lymphocytes in the pathogenesis of certain allergic diseases and autoimmune diseases such as Bronchial Asthma and hemorrhagic rectocolitis. But little data exist on the enzymatic activity of secretory granules associated with lymphocytes of patients suffering from these inflammatory diseases. The aim of the study was to characterize the activity of the DNase in the secretory granules of T lymphocytes isolated from peripheral blood of patients with Bronchial Asthma (n = 20) and Hemorrhagic Rectocolitis (n = 20). Thus, the secretory granules were isolated from lymphocytes by the ultracentrifugation method on percoll density gradient. The detection of the activity of the protein extracts was performed by zymography and electrophoresis method. The results reveal the presence of a protein extract with a molecular weight of 66 kDa both at the level of the granules of the lymphocytes of patients suffering from hemorrhagic rectocolitis considered as a classical autoimmune disease and in the granules of lymphocytes of patients with Bronchial Asthma. The study of physicochemical properties showed an increase in the enzymatic activity of DNase of secretory granules when 1 mM Ca2+ was added to the incubation medium at a pH = 7.5. On the other hand, the addition of 1 mM Zn2 + causes the inhibition of enzyme activity. These results suggest that the enzymatic activity of DNase detected in the granule of lymphocytes of patients with Bronchial Asthma and hemorrhagic rectocolitis occurs not only in the fragmentation of double stranded DNA but could also play a role in the apoptotic process of these same T-lymphocytes. The study of the properties of this DNase in the inflammatory diseases could enable to use this protein as a marker for determining the severity of disease.

Keywords

References

[1]  Ярилин, А.А. Base of Immunology. – М.: Меdicine, 1999. 608 с.
 
[2]  Green, D.R. Ferguson, L. Zitvogel, G. Kroemer. Immunogenic and tolerogenic cell death. Nat Rev Immunol 2009; 9: 353-363.
 
[3]  Elmore, E. Apoptosis: a review of programmed cell death. Toxicol Pathol 2007; 35: 495-516.
 
[4]  Rubinsztein, D.C, Williams A, S. Sarkar, et al. Novel targets for Huntington's disease in an mTOR-independent autophagy pathway. Nat Chem Biol 2008; 4 (5):295-305.
 
[5]  Косарев, В.В , Лотков B.C, Куклин A.C et al. Ингаляционные кортикостероиды в терапии бронхиальной астмы. Терапевт, арх 2000; 8 : 59-61.
 
Show More References
[6]  Spinozzi, F, Benedictis F.M. Apoptosis, airway inflammation and anti-asthma therapy: from immunobiology to clinical application. Pediatr Allergy Immunol 2008; 19 (4): 287-295.
 
[7]  Vodounon AC, Ckibo Y, Aikou N, Akpona S, Abramova ZI, Kotchoni OS,Baba-Moussa L. Morphological and biochemical characteristics of apoptosis lymphocytes of peripheral blood in the pathogenesis of atopic bronchial asthma of light and serious severity. Trade Sci Inc (Res. Rev BioSci) 2012; 6(8): 210-20.
 
[8]  Pio, R, Gonzalez A, Lopez-Zabalza et al. Granule associated DNase in T4 and T8 lymphocytes from patients with autoimmune diseases. Biochimica et Biophysica Acta 1998; 1406: 51-61.
 
[9]  Parrish, J.Z, Deng X. Cuts can kill: the roles of apoptotic nucleases in cell death and animal development. Chromosoma 2006; 115: 89-97.
 
[10]  Patel D, Rubbi CP, Rickwood D. Separation of T and B lymphocytes from human peripheral blood mononuclear cells using density perturbation methods. Clin Chim Acta 1995; 240: 187-93.
 
[11]  Ulmer, A.J, Scholz W, Ernest M et al. Isolation and subfractionation of human peripheral blood mononuclear cells (PBMC) by density gradient centrifugation on Percoll. Immunobiology 1984;166: 238-50.
 
[12]  Boyum, A, Brincker-Fjerdingstad I et al. Separation of human lymphocytes from citrated blood by density gradient (NycoPrep) centrifugation: monocyte depletion depending upon activation of membrane potassium channels. Scand J Immunol 2002; 56: 76-84.
 
[13]  F.A. Khorsid F.A and S.S. Mushref S.S. In vitro Anticancer Agent I-Tissue Culture Study of Human Lung Cancer Cells A549 II-Tissue Culture Study of Mice Leukemia Cells L1210. International Journal of Cancer Research 2006; 2: 330-344.
 
[14]  Borregaard N, Heiple J.M, Simons E.R, Clark R.A. Subcellular localization of the b-cytochrome component of the human neutrophil microbicidal oxidase: Translocation during activation. J. Cell Biol 1983; 97: 52-61.
 
[15]  Podack ER, Konigsberg PJ. Cytolytic T cell granules. Isolation, structural, biochemical, and functional characterization. J Exp Med 1984; 160: 695-710.
 
[16]  Thiery, J.,Walch M, Jensen D.K, Jensen D. Isolation of Cytotoxic T Cell and NK granules and purification of their effector proteins. Current Protocols in Cell Biology 2010; 3:37(1)3.37.29.
 
[17]  Laemmli, U.K. Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4. Nature 1970; 227: 680-685.
 
[18]  Napirei, M, Jensen A, Eulitz D et al. Expression pattern of the deoxyribonuclease 1 gene: lessons from the Dnase 1 knockout mouse. Biochem J. 2004; 380(Pt 3): 929-337.
 
[19]  Urboniene, D, Sakalauskas R, Sitkauskiene B. Autoimmunity in pathogenesis of chronic obstructive pulmonary disease. Medicina (Kaunas) 2005;4 (13): 190-195.
 
[20]  Kopinski, P, Przybylsk G, Balicka-Slusarczyk et al. Apoptosis of alveolar lymphocytes in sarcoidosis and in control group is more frequentin smokers then in nonsmoking persons. Przegl Lek. 2006; 63: 841-847.
 
[21]  Vignola, A.M, Chanez P, . Chiappara et al. Evaluation of apoptosis of eosinophils, macrophages, and T lymphocytes in mucosal biopsy specimens of patients with asthma and chronic bronchitis. J Allergy Clin Imunol 1999; 103:563-573.
 
[22]  Busse, W.W, Lemanska R.F. Аsthma. NEJM 2001; 344:350-362.
 
[23]  Мамонтова, Т.В, Kaidathev I.P. Новые аспекты апоптоза мононуклеарных клеток в патогенезе атопической бронхиальной астмы. Аllergology 2005;4: 15-23.
 
[24]  Chau-Ching, L, Young L.H.Y et al. Lymphocyte-mediated cytolysis and disease. N. Engl. J. Med; 1996; 335: 1651-1659.
 
Show Less References

Article

Drug resistant Staphylococcus aureus in Clinical Samples at Kampala International University-teaching Hospital, Bushenyi District, Uganda

1School of Pharmacy, Kampala International University-Western Campus. P.O BOX 71, Bushenyi, Uganda

2Department of Biochemistry, Kampala International University-Western Campus. P.O BOX 71, Bushenyi, Uganda

3Department of Microbiology & Immunology, Kampala International University-Western Campus. P.O BOX 71, Bushenyi, Uganda

4School of Pharmacy and Health Sciences, United States International University – Africa, Nairobi, Kenya


American Journal of Biomedical Research. 2016, 4(4), 94-98
doi: 10.12691/ajbr-4-4-3
Copyright © 2016 Science and Education Publishing

Cite this paper:
Janet Nalwoga, Michael Tirwomwe, Albert Nyanchoka Onchweri, Josephat Nyabayo Maniga, Cyprian Mose Nyaribo, Conrad Ondieki Miruka. Drug resistant Staphylococcus aureus in Clinical Samples at Kampala International University-teaching Hospital, Bushenyi District, Uganda. American Journal of Biomedical Research. 2016; 4(4):94-98. doi: 10.12691/ajbr-4-4-3.

Correspondence to: Conrad  Ondieki Miruka, Department of Biochemistry, Kampala International University-Western Campus. P.O BOX 71, Bushenyi, Uganda. Email: conradmiruka@yahoo.com

Abstract

Background: Staphylococcus aureus that is resistant to methicillin is an important nosocomial pathogen that often causes infections that are hard to treat. This is due to the fact that the pathogen is usually resistant to other commonly used antibiotics. The presence of drug resistant MRSA among patients has previously been documented in various parts of Uganda. However no reports have been documented for Kampala International University-Teaching hospital in western Uganda. This study was therefore carried out to determine the prevalence and antibiotic resistance of MRSA strains in the patients hospitalized in the surgical ward. Methods: Wound swabs were collected from both male and female patients hospitalized in the surgical ward. Samples were then cultured in suitable media. The Staphylococcus aureus colonies that were obtained were tested for resistance to oxacillin to determine the strains that were MRSA. Further antibiotic resistance of the MRSA isolates was determined by the disc diffusion method using various antibiotics. Results: Out of the one hundred and fourteen isolates from the clinical samples, only seventy five isolates were clearly identified as S. aureus with 85.3% coagulase positive and 13.3% coagulase negative. Methicillin resistant staphylococcus aureus was prevalent at a rate of 56.1%. among the MRSA isolates, resistance to ciprofloxacin was observed to be the highest while resistance to ceftriaxone was observed to be the least. Conclusion: The high prevalence of MRSA amongst the surgical ward patients requires proper measures to be taken to prevent further spread of the pathogen. It is recommended that the source of this drug resistant strains of MRSA be determined so as to design appropriate interventions to prevent the future emergence of infections that are hard to treat.

Keywords

References

[1]  Appelbaum PC. “MRSA—the tip of the iceberg”. Clin Microbiol Infect. 12(Suppl 2): 3-10. 2006.
 
[2]  Brown DF, Edwards DI, Hawkey PM, et al, on behalf of the Joint Working Party of the British Society for Antimicrobial Therapy, Hospital Infection Society, Infection Control Nurses Association. “Guidelines for the laboratory diagnosis and susceptibility testing of methicillin- resistant Staphylococcus aureus (MRSA)”. J Antimicrob Chemother 56: 1000-18. 2005.
 
[3]  Francis JS, Doherty MC, Lopatin U, Johnston CP, Sinha G, Ross T et al. “Severe community-onset pneumonia in healthy adults caused by methicillin-resistant Staphylococcus aureus carrying the Panton-Valentine leukocidin genes”. Clin Infect Dis. 40: 100-7. 2005.
 
[4]  Healy CM, Hulten KG, Palazzi DL, Campbell JR, Baker CJ. “Emergence of new strains of methicillin-resistant Staphylococcus aureus in a neonatal intensive care unit”. Clin Infect Dis. 39: 1460-6. 2004
 
[5]  Saxen S, Singh K. and Talwar V. “Methicillin Resistance Staphylococcus aureus prevalence in Community in East Delhi area”. Jpn J Infect Dis. 56: 54-56. 2003
 
Show More References
[6]  Ojulong J, Mwambu TP, Joloba M, Bwanga F, Kaddu-Mulindwa DH. “Relative prevalence of methicillin resistant Staphylococcus aureus and its susceptibility pattern in Mulago Hospital, Kampala, Uganda”. Tanzan J Health Res 11: 149-153. 2009
 
[7]  Moremi N, Mshana SE, Kamugisha E, Kataraihya J, Tappe D, et al. “Predominance of methicillin resistant Staphylococcus aureus -ST88 and new ST1797 causing wound infection and abscesses”. J Infect Dev Ctries 6: 620-625. 2012.
 
[8]  Ojikan-Odeke. M.Med. dissertation on “Hand infections in Uganda”. 19-40. 1978.
 
[9]  Mugisa D.B. M. Med Surgery dissertation on “Complications following laparotomy in Mulago Hospital”. 59-156. 1988.
 
[10]  Olaro .C. M.Med Surgery dissertation on “Study to assess risk factors for Postoperative Complication following abdominal Surgery in Mulago hospital”. 1-3. 1999.
 
[11]  Kitara LD, Anywar AD, Acullu D, Odongo-Aginya E, Aloyo J, Fendu M. “Antibiotic susceptibility of Staphylococcus aureus in suppurative lesions in Lacor Hospital, Uganda”. African Health Sciences. 11 (S1): S34 - S39. 2011.
 
[12]  Shanson DC. “Antibiotic Resistant Staph aureus”. J Hospit infect. 2:11-16. 1981.
 
[13]  Boyce JM. “Increasing prevalence of MRSA in the USA”. J Infect Control and Hospital Epidemiol. 11:639-642. 1990.
 
[14]  Kateete D, Kimani C, Katabazi F, Okeng A, Okee M, Nanteza A, Joloba M, Najjuka F. “Identification of Staphylococcus aureus: DNase and Mannitol salt agar improve the efficiency of the tube coagulase test”. Annals of Clinical Microbiology and Antimicrobials 9(1):23. 2010.
 
[15]  Bull AL, Worth LJ, Richards MJ. “Impact of Vancomycin Surgical Antibiotic Prophylaxis on the Development of Methicillin-Sensitive Staphylococcus aureus Surgical Site Infections: Report From Australian Surveillance Data (VICNISS)”. Annals of surgery. 256 (6): 1089-1092. 2012.
 
[16]  Kateete DP, Namazzi S, Okee M, Okeng A, Baluku H, Musisi NL, et al. “High prevalence of methicillin resistant Staphylococcus aureus in the surgical units of Mulago hospital in Kampala, Uganda”. BMC Research Notes. 4: 326. 2011.
 
[17]  J Ojulong, T Mwambu, M Jolobo, E Agwu, F Bwanga, C Najjuka, D Kaddu-Mulindwa. “Prevalence of Methicillin resistant Staphylococcus aureus (MRSA) among isolates from surgical site infections in Mulago hospital- Kampala, Uganda”. The Internet Journal of Infectious Diseases. Volume 7 Number 2. 2008.
 
[18]  Jean-Marie Liesse Iyamba, José Mulwahali Wambale, Cyprien Mbundu Lukukula, Ntondo za Balega Takaisi-Kikuni. “High prevalence of methicillin resistant staphylococci strains isolated from surgical site infections in Kinshasa”. Pan African Medical Journal. 18: 322. 2014.
 
[19]  Bashir Mwambi, Jacob Iramiot, Freddie Bwanga, Marthae Nakaye, Herbert Itabangi and Joel Bazira. “Clindamycin Resistance among Staphylococcus Aureus Isolated at Mbarara Regional Referral Hospital, in South Western Uganda”. British Microbiology Research Journal 4(12): 1335-1344. 2014.
 
[20]  Jeremiah Seni, Christine F Najjuka, David P Kateete, Patson Makobore, Moses L Joloba, Henry Kajumbula, et al. “Antimicrobial resistance in hospitalized surgical patients: a silently emerging public health concern in Uganda”. BMC Research Notes 6:298. 2013.
 
[21]  Najat Buzaid, Abdel-Naser Elzouki, Ibrahim Taher, Khalifa Sifaw Ghenghesh. “Methicillin-resistant Staphylococcus aureus (MRSA) in a tertiary surgical and trauma hospital in Benghazi, Libya”. J Infect Dev Ctries. 5(10): 723-726. 2011.
 
[22]  Ching Jou Lim, Allen C. Cheng, Jacqueline Kennon, Denis Spelman, Dayna Hale, Gabrielle Melican et al. “Prevalence of multidrug-resistant organisms and risk factors for carriage in long-term care facilities: a nested case–control study”. J Antimicrob Chemother 2014.
 
[23]  Meng Xiao, He Wang, Ying Zhao, Lei-Li Mao, Mitchell Brown, Yun-Song Yu et al. “National Surveillance of Methicillin-Resistant Staphylococcus aureus in China Highlights a Still-Evolving Epidemiology with 15 Novel Emerging Multilocus Sequence Types”. Journal of Clinical Microbiology 51 (11): 3638-3644. 2013.
 
[24]  Alain C. Juayang, Gemma B. de los Reyes,April Joy G. de la Rama, Christine T. Gallega. “Antibiotic Resistance Profiling of Staphylococcus aureus Isolated from Clinical Specimens in a Tertiary Hospital from 2010 to 2012”. Interdisciplinary Perspectives on Infectious Diseases 898457: 4. 2014.
 
[25]  Obianuju Onelum, Babatunde Odetoyin, Anthony Onipede and Adesola Oyelese. “The Role of Methicillin-Resistant Staphylococcus aureus in Clinical infections in Obafemi Awolowo University Teaching Hospitals Complex, Ile-Ife, South Western Nigeria”. Journal of Microbiology & Experimentation. 2(2): 00041. 2015.
 
[26]  Rita Khanal, Prakash Sah, Pramila Lamichhane, Apsana Lamsal, Sweety Upadhaya and Vijay Kumar Pahwa. “Nasal carriage of methicillin resistant Staphylococcus aureus among health care workers at a tertiary care hospital in Western Nepal”. Antimicrobial Resistance and Infection Control. 4:39. 2015.
 
[27]  Cosgrove SE, Qi Y, Kaye KS, Harbarth S, Karchmer AW, Carmeli Y. “The impact of methicillin resistance in Staphylococcus aureus bacteremia on patient outcomes: mortality, length of stay, and hospital charges”. Infect Control Hosp Epidemiol. 26: 166-74. 2005.
 
[28]  Gastmeier P, Schwab F, Sohr D, Behnke M, Geffers C. “Reproducibility of the surveillance effect to decrease nosocomial infection rates”. Infect Control Hosp Epidemiol. 30: 993-9. 2009.
 
[29]  G. Sganga, C. Tascini, E. Sozio, M. Carlini, P. Chirletti, F. Cortese et al. “Focus on the prophylaxis, epidemiology and therapy of methicillin-resistant Staphylococcus aureus surgical site infections and a position paper on associated risk factors: the perspective of an Italian group of surgeons”. World Journal of Emergency Surgery 11: 26. 2016.
 
[30]  Rubin RJ, Harrington CA, Poon A, Dietrich K, Greene JA, Moiduddin A. “The economic impact of Staphylococcus aureus infection in New York City hospitals”. Emerg Infect Dis. 5: 9-17. 1999.
 
[31]  Pandya N, Chaudhary A, Mehta S, Parmar R. “Characterization of Methicillin Resistant Staphylococcus aureus from various clinical samples at Tertiary care hospital of rural Gujarat”. J Res Med Den Sci. 2(3): 49-53. 2014.
 
Show Less References

Article

Cloning Open Reading Frame (ORF) of Rv2430c Mycobacterium tuberculosis Indonesian Isolate in Escherichia coli JM 109

1Department Of Biology, Faculty of Life Sciences, Hasanuddin University, Indonesia

2Department of Microbiology, Faculty of Medicine, Hasanuddin University, Indonesia

3Development Ministry of Health of Republic of Indonesia, Central Basic Biomedical, Health Technology National Institute of Health and Research, Indonesia


American Journal of Biomedical Research. 2016, 4(4), 99-101
doi: 10.12691/ajbr-4-4-4
Copyright © 2016 Science and Education Publishing

Cite this paper:
Rosana Agus, Muhammad Nasrum Massi, Francisca Srioetami Tanoerahardjo. Cloning Open Reading Frame (ORF) of Rv2430c Mycobacterium tuberculosis Indonesian Isolate in Escherichia coli JM 109. American Journal of Biomedical Research. 2016; 4(4):99-101. doi: 10.12691/ajbr-4-4-4.

Correspondence to: Rosana  Agus, Department Of Biology, Faculty of Life Sciences, Hasanuddin University, Indonesia. Email: rosana_agus@yahoo.com

Abstract

Various strategies have been implemented to prevent tuberculosis. Vaccination with Bacille Calmette Guerin (BCG) vaccine is still used around the world. Generally, most people in have gained BCG vaccine as an infant, but the effectiveness of these vaccines do not survive to adulthood. Therefore, the necessary replacement BCG vaccine more effective to eliminate tuberculosis. Mycobacterium tuberculosis is an intracellular pathogen and it was inside the macrophage, which is considered to be the most important component of the immune system. M. tuberculosis has two sets of genes are highly polymorphic referred to as PE and PPE families. These unique families of proteins account for about 10% of the mycobacterial genome and have attracted great interest from a variety of different studies around the world. One member of the as a vaccine candidate is Rv 2430c. It is known that the sera of all patients infected with TB showed strong antibody responses against Rv 2430c compared to healthy individuals. The existence of these antibodies indicates that this protein is found in vivo during infection and is a native immunogenic molecule. The purpose of this study was to clone the Open Reading Frame (ORF) Rv 2430c M. tuberculosis Indonesian isolates to host cells Esherichia coli JM 109. The method used is by isolating -chromosomal DNA from clinical isolates from Indonesia, amplifying the ORF Rv 2430c with , ligating into cloning vectors pGEM-T and transform to E.coli JM 109. Characterization of clones do with migration analysis, restriction analysis and . The results obtained are recombinant clones that carry insertion was Rv 2430cKeywords: Mycobacterium tuberculosis, Rv 2430c, ORF, , JM 109.

Keywords

References

[1]  World Health Organization. Global tuberculosis report, World Health Organization, Geneva, 2013.
 
[2]  Abubakar, I., L. Pimpin, C. Ariti, R. Beynon, P. Mangtani, J. A. Sterne, P. E. Fine, P. G. Smith, M. Lipman, D. Elliman, et al. Systematic review and meta-analysis of the current evidence on the duration of protection by bacillus Calmette-Gue´rin vaccination against tuberculosis. Health Technol. Assess.17: 1-372, v–vi, 2013.
 
[3]  Hart, P. D., and I. Sutherland. BCG and vole bacillus vaccines in the prevention of tuberculosis in adolescence and early adult life. BMJ 2: 293-295, 1977
 
[4]  Baily, G. V. 1980. Tuberculosis prevention Trial, Madras. Indian J. Med. Res. 72 (Suppl.): 1-74, 1980.
 
[5]  Brennan, M. J., and J. Thole. Tuberculosis vaccines: a strategic blueprint for the next decade. Tuberculosis (Edinb.) 92(Suppl. 1): S6-S13, 2012.
 
Show More References
[6]  Checkley, A. M., and H. McShane. Tuberculosis vaccines: progress and challenges. Trends Pharmacol. Sci. 32: 601-606, 2011.
 
[7]  Choudhary R.K, Sangita Mukhopadhyay, Prachee Chakhaiyar, Naresh Sharma, K.J.R Murthy, V.M Katoch and Seyed E. Hasnain, PPE antigen Rv 2430 c of Mycobacterium tuberculosis induces a strong B- cell Response, Infection and Immunity, Vol 71 No 11, p.6338-6343, 2003.
 
[8]  Sambrook, J., Fristch, E.F. dan Maniatis, T. Molecular Cloning A Laboratory Manual. Cold Spring Harbor Laboratory Press, 1989.
 
[9]  Jean and Alexander Heard Library, 2016, BSCI 1510l: Detecting uncut Plasmids from the Restriction Digests.
 
Show Less References