Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-5241210EnglishN-0001November30HealthcareEFFECTIVENESS OF PASSIVE STRETCHING VERSUS HOLD RELAX TECHNIQUES IN FLEXIBILITY OF
HAMSTRING MUSCLE
English0308Gauri ShankarEnglish Yogita AchnaniEnglishAim: To compare the effectiveness of passive stretching and hold relax techniques in the flexibility of hamstring muscle.
Methods: A total of 80 normal healthy female subjects between age group 20-30 years referred to the department of physiotherapy, Sumandeep Vidyapeeth University, sampling method being convenient sampling. The subjects were randomly divided in two groups i.e. passive stretching group (n=40) and Proprioceptive Neuromuscular Facilitation (PNF) group (n=40) and given passive stretching and proprioceptive neuromuscular facilitation technique respectively. Active knee extension range was measured before and after the intervention by goniometer.
Results: The t test showed a highly significant (p=0.000) increase in range of motion in Proprioceptive Neuromuscular Facilitation (PNF) group.
Conclusion: Proprioceptive neuromuscular facilitation (PNF) technique is more effective in increasing hamstring flexibility than the passive stretching.
EnglishStretching, Flexibility, PNF, Hold-relax, Hamstring.INTRODUCTION
Flexibility is a key component for prevention of injury and rehabilitation, stretching decreases injury and improves performance in sports for overall fitness.1 Stretching is a therapeutic manoeuvre designed to increase mobility of soft tissue and subsequently improves range of motion by elongating structures that have adaptively shortened and have become hypomobile over time.2 Proprioceptive neuromuscular facilitation (PNF) is used as inhibition technique to assist with muscle elongation. 3 Passive stretching and isometric contraction encourage flexibility or coordinate throughout limbs range of motion. Proprioceptive neuromuscular facilitation is used to supplement daily stretching when employed to quick gain in range of motion it decreases fatigue and prevents overuse injuries.4
A hold relax is a technique of facilitating normal muscle sensation and muscle awareness, used in treating hyper tonicity or motor dysfunction. It is often applied when there is muscle tightness in one side of joint and when immobility is the result of pain.5 It is a relaxation technique to obtain a lengthening reaction of muscle whose action is antagonistic to movement limited in range, it means increasing range of movement in joints, it is effective, simple and pain free. 3 Stretching recommendations are clouded by many misconceptions and conflicts. Research reports despite being limited has been promoted for years as an integral part of fitness programme to decrease risk of injury and relieve pain associated with sports.6 The specificity of movement that a person performs in regular physical activity and stretching method often define the development and improve body?s range of motion. Goal of all stretching programme is to provide joint mobility while maintaining joint stability.7
Muscle tightness is a limiting factor for optimal physical performance and an important intrinsic factor for sports injury. 8 Hamstring as one of the commonest muscles often gets tight as the biomechanics of hamstrings are complex because they pull over two joints - hip and knee. Therefore, their efficacy in producing force at knee is dictated by the angle of hip joint. Greater hamstring force is produced with hip in flexion when hamstring is lengthened over joint, regardless of knee position.1 Tight hamstrings can have profound effect on seated postural alignment. Posterior pelvic tilt is a primary effect, with resultant kyphotic thoracic spine and stress on cervical spine either hyper extended or flexed. If hamstrings are unequally tight, the pelvis will rotate away from tighter hamstrings. For example; if right hamstrings are tighter, the pelvis will rotate towards left.9 Reduced hamstring muscle flexibility has been implicated in lumbar spine dysfunction, with number of studies showing positive correlation between decreased hamstrings, flexibility and low back pain.10
METHODOLOGY A total of 80 asymptomatic subjects among student community of Sumandeep Vidyapeeth University, Baroda, within 20-30 years of age group were included in this study who met the inclusion and exclusion criteria.
Inclusion criteria:
Age- 20 to 30 years
Sex – Females
Exclusion criteria:
Orthopedic problems
Neurological conditions
Psychological disorders
Instrumentation:
Double arm goniometer
Stop watch
Pen
Pencil and paper
PROCEDURE In this study, 80 subjects were included within age group 20-30 years who fulfilled inclusion criteria. Before starting intervention, active knee extension range in high sitting position with hip in 90 degree flexion was measured through goniometer. After completion of study, active knee extension range was measured again in the same way. Subjects were equally divided in two groups as follows.
Group 1: Passive Stretching Group which was given passive stretching Procedure: Investigator knelt down on the mat and placed patient?s heel or distal tibia against own shoulder and placed both of hands along the anterior aspect of distant thigh to keep knee extended, opposite extremity is stabilized in extension by belt and held in place by therapist?s knee with in 0 degree extension, hip in neutral rotation and then investigator flexes the hip as far as possible.
Group 2: PNF Group which was given Hold Relax technique Procedure: Subjects were randomly assigned and modified hold relax stretch performed with hip in neutral position. For each stretching, investigator gave passive stretching until the subject reported mild stretching sensation and held that position for 7 seconds, next, sub maximal isometric contraction of hamstring muscle for 7 seconds by asking the subject to push her leg back towards the table against the resistance of investigator after contraction relaxation for 5 seconds. Investigator then passively stretched the muscle until the mild stretch sensation was reported the stretch was held for 7 seconds. This sequence was repeated 5 times on each subject.
RESULT ANALYSIS
In intergroup comparison there is increase in range of motion (73.650 ) after passive stretching in Group 1 and range of motion (ROM) increased after hold relax (79.320 ) than before hold relax (73.050 ) in Group 2.
DISCUSSION The current study is a comparative study done with an attempt on increasing the hamstring length by using 2 different techniques, i.e. passive stretching and hold relax. In this study, 80 normal female subjects have been taken and randomly divided into 2 groups. Active knee extension is measured using goniometer before any physical intervention and at the end of intervention it is measured again. The study shows highly significant increase in ROM in both groups but this finding cannot be generalised or converted to clinically significant data since sample of male population in current study is not included, while comparing both groups, hold relax is found more effective than passive stretching (t-value -4.763). Hold relax has been found to be more effective because the isometric muscle action completed immediately before the passive stretching which helps to achieve autogenic inhibition - a reflex relaxation that occurs in muscle where the golgi tendon organ is stimulated. The present finding is in accordance with “Scott Spernoga et al, who found that sequence of 5 modified hold relax stretching produced significant increase in hamstring flexibility.11 On the basis of this study, modified hold relax protocol has been used and the present study shows that hold relax increases more range of motion11, this is consistent with “Sharman Malanie” who found that proprioceptive neuromuscular facilitation produces superior ROM12 .
Our current study is not in accordance with “Worrell TW et al. (1994), who found that 60 seconds stretching had no improvement in ROM as for ROM to be maintained, stretching must be continued13 and “Sullivan Mk, et al. (1992) who concluded that there is no significant increase in hamstring flexibility with either of stretching technique.14 There is another study which shows that there is no significant difference in ROM in standing and supine hamstring stretching as they are equally effectively conducted by “Decoster LC (2004). 15
Certain variables are uncontrolled in the study and their influence on the results is assumed to be null, the result affected by assumption which is different level of physical activity of subjects. The difference in the force applied for stretching to the subjects may be confounding factor in result obtained.
CONCLUSION It is conclude that hold relax a technique of proprioceptive neuromuscular facilitation is more effective in increasing hamstring flexibility than passive stretching.
SUGGESTIONS The limitations of the current study were the sample size and female population. Though the result obtained was very highly significant to generalize the findings into a clinically useful data the study has to be replicated in a bigger sample size. If the uncontrolled variables of the current study could be controlled, the sensitivity and specificity of the findings can be increased. Further studies should also aim at studying both the genders separately to obtain a more accurate and generalized results
Englishhttp://ijcrr.com/abstract.php?article_id=2212http://ijcrr.com/article_html.php?did=22121. Corbin C B Noble, Flexibility – a major component of physical fitness, Journal of Physical Education, 1980; 51-57.
2. Carolyn Kisner, Lynn Allen Colbey : Therapeutic Exercise Foundations And Techniques, Fourth Edition, FA Davis Company 2003,173
3. M Dena Gardiner, Principles of Exercise Therapy ,Fourth Edition, CBS Publication 2000,89
4. MC Atee, Robert , Facilitating Stretching, Third Edition 2007,Human Kinetics,6:9
5. Mosby?s medical dictionary, Ellsevier, Eighth Edition
6. Birgit Schuback, Julie Hooper, A Comparison of self stretching incorporating proprioceptive neuromuscular facilitation components and therapist applied proprioceptive neuromuscular facilitation technique on hamstring flexibility, Journal of Physiotherapy,2004, 90:51-57.
7. Len Karvitz, P.H.D., Vivian H Heyward, Flexibility Training Journal of Fitness Training 2000, 7:18-20
8. Pamela K Levangie, Cynthia C. Norkin, Joint Structure and Function, Fourth Edition, FA Davis Company 2005, 376
9. Caroline Portoghese ,Rehabilitation Management, The interdisciplinary journal of rehabilitation, june 2005
10. S.James ,D Joshua, Lengthening the hamstrings muscles without stretching using „Awareness through movement? A journal of physical therapy 2006,86
11. Scott .G. Spernoga, Tinothy L Uhl, Duration maintained hamstrings flexibility after one time, modified hold relax stretching protocol, Journal of Athletic Training,2001, 6: 44-48.
12. Sharmen Malanie , Proprioceptive neuromuscular facilitation stretching Mechanism and clinical implication. Journal of Sports Medicine 2006 ,36 (11)929-939.
13. Worrell TW, Smith TL, et al, Effect of hamstring stretching on hamstring muscle performance, Journal of orthopaedic sports 1994 sep,20 (3), 154-159.
14. Sullivan MK, Dejullian JJ et. All, Effect of pelvic position and stretching method on hamstring muscle flexibility. Journal of Medical Science ,Sports exercise, 1992,24 : 1383-1389.
15. Decoster LC, Standing and Supine hamstring stretching are equally effective, Journal of Athletic training 2004, Dec 39(4); 330-334.
Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-5241210EnglishN-0001November30TechnologyCLUSTERING OF DATA AFTER MINIMIZING DATA SIZE USING ROUGH SET THEORY
English0915Sunanda DasEnglish Asit Kumar DasEnglishObjective: Our approach is to reduce the large data size to a small data size which
represents same features of the total large data set, so that computational complexity
becomes shorter.
Method: In this paper we present a new approach to minimize the data size and then to
cluster that reduced data. The volume of data being generated nowadays to cluster is
increasingly large. How to extract useful information from such data collections is an
important issue. A promising technique is the Rough set theory, a new mathematical
approach to data analysis based on objects of interest into similarity classes which are
indiscernible with respect to some features.
Result and conclusion: This theory offers two fundamental concepts: reduct and core.
In this paper, some basic ideas of rough set theory are first presented. Some experiment
results are also given.
EnglishRough set theory, Data mining, correlationINTRODUCTION
Data mining is an emerging area of computational intelligence that offers new theories, techniques, and tools for processing large volumes of data. It has gained considerable attention among practitioners and researchers as evidenced by the number of publications, conferences, and application reports. The growing volume of data that is available in a digital form has accelerated this interest. Data mining relates to other areas, including machine learning, cluster analysis, regression analysis, and neural networks [1-5]. Rough set theory [6] is a relatively new mathematical technique developed by Pawlak in the 1980s to describe quantitatively uncertainty, imprecision and vagueness. Classical set theory deals with crisp sets and rough set theory may be considered an extension of the classical set theory. The rough set approach has many advantages. An important step in the knowledge discovery process is the reduction of thedimensionality of data. In real database systems, though there are many attributes and records, in some circumstances, in fact only some of the attributes are indispensable. If the dispensable attributes can be eliminated, the complexity of analyzing the data can be greatly reduced. Our algorithm is based on rough set theory which consists of two parts. The first part is for attribute reduction and the second is for rule extraction.
Dataset: In this experiment we use a wine dataset having 13 different attributes each of having 178 different data values. Using the 10 fold method we divide this dataset into 10 different test datasets and train datasets.
MATERIALS AND METHODS
Correlation:
Using correlation function in MATLAB, we get 10 different tables for 10 different train datasets in which each attribute value shows the correlation with other attributes.
RESULT AND DISCUSSION
After doing the stepwise experimental work, we get 10 correlation matrix. From the correlation-matrix of first training dataset, we got graph as given in Figure 1. The correlation coefficients are a normalized measure of the strength of the linear relationship between two variables and range between -1 and 1, where -1 means that one column of data has a negative linear relationship to another column of data. 0 means there is no linear relationship between the data columns. 1 means that there is a positive linear relationship between the data columns. Figure 1 is a grouped bar graph. The bars in the first group correspond to the first row of the matrix, the 2nd group to the 2nd row and so on. In this figure, 1st group bar, there are 13 bars each of which represents the correlation between each pair of attribute ( row & column). which is having negative and positive values in correlation are represented in both side of the origin, in such a way that each cluster of column bar shows 13 attributes in positive and negative value. So, this above Figure1 represents the graphical representation of correlation-matrix of first training dataset of wine dataset. After getting the correlation values between the 13 attributes of wine dataset, we get the 8 different Functional Dependencies using the threshold value θ = 0.5. And then using closer property we get 7 different values to predict the classification of these attributes i.e. {A,J}, {F,G,L,M,I,K}, {B,C,D,E,H}. Then using the Information gain formula, we find that the set{ F,G,L,M,I,K } and {B,C,D,E,H} can not be clustered again. Then using cardinality formula, the 3 different cardinality values in percentage of the sets {A,J}, {F,G,L,M,I,K}, {B,C,D,E,H} are generelated that are 32.9%, 68.4% , 42.7% respectively. Among these values 68.4% of set {F,G,L,M,I,K} is the highest value. This means that the reduct set {F,G,L,M,I,K} only can represent the characteristics of the total 13 attributes of this wine dataset.
CONCLUSION
In this paper, basic concepts of data mining and the rough set theory were discussed. The patterns formed by the rules extracted with rough set theory differ from other patterns. Here the method was illustrated with a numerical example. This method shows that instead of handling large volume of data, we can easily work with small-size of data which gives same meaningful information and characteristics of the whole data. This method enhances the utility of the extracted knowledge, reduces timecomplexity. This method can be further enhanced by getting the cardinality value 100% approximately.
Englishhttp://ijcrr.com/abstract.php?article_id=2213http://ijcrr.com/article_html.php?did=22131. Andrew Kusiak. Rough Set Theory: A Data Mining Tool for Semiconductor Manufacturing. IEEE Transactions on Electronics Packaging Manufacturing 2001; 24(1):44-50. 14 International Journal of Current Research and Review www.ijcrr.com Vol. 02 issue 10 Oct 2010
2. Langley P, Simon HA. Applications of machine learning and rule induction. Commun. ACM 1995; 38(11) :55–64.
3. Carbonell JG. Machine Learning: Paradigms and Methods. J. G. Carbonell, Ed. Cambridge. MA: MIT Press 1990.
4. Reiter R. A theory of diagnosis form first principles. Artif. Intell 1987; 35: 57–95.
5. Barto A, Sutton RS. Reinforcement Learning. Cambridge. MA: MIT Press 1998.
6. Pawlak Z. Rough sets. Int. J. Inform. Comput.Sci 1982; 11(5): 341-356.
7. Pawlak Z. Rough Sets - Theoretical Aspects of Reasoning about Data. Kluwer Academic Publishers, Boston, London, Dordrecht 1991:229.
8. Pawlak Z. Rough set theory and its applications to data analysis. Cybernetics and Systems 1998; 29: 661-688 .
9. Jiawei Han, Micheline Kamber. Data Mining: Concepts and Techniques. Data Mining Books, Publisher: Elsevier Science Ltd. Second edition, China Machine Press :296 -303.
10. Abraham Silberschatz, Henry Korth, Sudarshan S. Database System Concepts, Database Books. McGraw-Hill.
11. Ihn-Han Bae, Hwa-Ju Lee, KyungSook Lee. Design and evaluation of a rough set- based anomaly detection scheme Considering weighted feature values. International Journal of KnowledgeBased and Intelligent Engg. System 2007; 11:201-206.
Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-5241210EnglishN-0001November30General SciencesCONSTRUCTION OF CHIMERIC CRY2A GENE OF BACILLUS THURINGIENSIS BY DOMAIN SWAPPING AND ITS ANALYSIS
English1626Indra Arulselvi PEnglish Udayasuriyan VEnglishA chimeric cry2A gene of Bacillus thuringiensis (Bt) was constructed by domain swapping. DNA fragment corresponding to domain I & II of Cry2Aa and domain III of Cry2Ac was amplified by PCR and cloned individually in a separate pBluescript vector. The cloned DNA fragments of cry2A genes were aligned to construct a chimeric cry2Ax2 gene. The chimeric cry2Ax2 gene was cloned in an E.coli-Bt shuttle vector in
between cry3Aa promoter and cry2Aa terminator, for gene expression studies. Though the presence of chimeric cry2Ax2 gene has been verified by PCR, its expression in the transformants of acrystalliferous Bt strain, 4Q7, was not obvious in SDS-PAGE analysis. The non-expression of the chimeric cry2Ax2 gene may be attributed to the biasness of the new codons introduced during the process of its construction. Expression of the newly constructed chimeric cry2Ax2 gene may be achieved in the recombinant Bt strain after site directed mutagenesis or by constructing the gene by a suitable promoter in E. coli. After getting the chimeric gene expressed, it can be used for further bioassays to assess its toxicity against lepidopteron pest.
EnglishBacillus thuringiensis, chimeric cry2A gene, domain swapping, E. coli-Bt shuttle vector.INTRODUCTION
Bacillus thuringiensis (Bt), is a wellknown gram-positive, spore-forming soil bacterium that forms parasporal insecticidal crystal proteins during the stationary phase of its growth cycle. These proteins are termed deltaendotoxins because of their intracellular location and have been used for many years as successful biological insecticides1 . Cloning of the first crystal protein gene (cry) of Bt was reported by Schnepf & Whiteley2 , since then more than 270 cry genes have been cloned, characterized, and their classification based on amino acid sequence similarity of their proteins3 . Cloning of cry genes provides an opportunity to express the cloned gene in acrystalliferous Bt or E. coli to find out the insecticidal activities of their proteins. In the natural isolates of Bt the cry2Aa and cry2Ac genes are expressed as third orf in operon model whereas cry2Ab gene is cryptic in nature. Dankocsik et al. 4 achieved expression of cry2Ab gene by the promoter of cry3Aa gene in an acrystalliferous strain of Bt. Transgenic crops that produce Cry1A toxin can control some key lepidopteran pests5 . Continuous exposure to a single kind of Bt toxin can lead to resistance development in insect pests. Routine replacement of cry genes or pyramiding of cry genes could be useful for effective control of insect pests by transgenic technology. The Cry2A proteins of B. thuringiensis are promising candidates for management of resistance development in insects due to its differences from the currently used Cry1A proteins, in structure6 and mode of action7 . Combination of Cry1Ac and Cry2Ab is used in the second version of Bt cotton (Bollgard® II) in USA and Australia. But the Indian populations of H. armigera were thirtyfive fold less susceptible to Cry2Aa than Cry1Ac8 . Studies are insufficient on susceptibility of Indian population of H. armigera to different Cry2A proteins. Variation of a single amino acid can significantly influence the level of toxicity in Cry proteins9,10 . New variants of the already known cry gene subgroups11 , mutated12,13 and chimeric13 cry gene sequences could encode crystal proteins with significant difference in the level of toxicity due to variation in their sequences. Hence, in the present study a new chimeric cry2A gene was constructed and analysed.
MATERIALS AND METHODS
Genomic DNA isolation and amplification of Cry2A domains
Plasmid DNA was isolated from recombinant E. coli strains, harbouring p2Aa and p2Ac plasmids. The p2Aa plasmid contains cry2Aa operon and p2Ac plasmid contains cry2Ac operon. The plasmid DNA isolated from p2Aa and p2Ac plasmids was used as a template for PCR amplification. DNA fragments of ~1465 bp and~441 bp was amplified using 2AFS & 2AR2C and 2CF2C & 2ARS primers (Table 1). The PCR was performed for 30 cycles as follows: 94 °C for 1 min, 60 °C for 45 sec and 72 °C for 1.0 min., the final extension was performed for 7 min at 72 °C. The amplicons were column purified and examined on 1.2 % agarose gel.
Cloning of amplicons individually in pBKS vector Two sets of pBKS vector
were linearized by double digestion with KpnI and HindIII for one set and HindIII and XbaI for another set. The amplicon of ~1465 bp (domain I & II of Cry2Aa) was double digested with KpnI and HindIII. The amplicon of ~441 bp (domain III of Cry2Ac) was also double digested with HindIII and XbaI. The digested vector and the amplicons were resolved in agarose gel, excised from the gel and purified by gel extraction kit. Two sets of ligation mixture were prepared. The pBluescript vector digested with KpnI and HindIII
restriction enzymes and the insert DNA fragment of ~1465 bp digested by the same enzymes were used in one set of ligation. In another set of ligation mixture, the pBluescript vector digested with HindIII and XbaI restriction enzymes and the insert DNA fragment of ~441 bp digested by the same enzymes were used. The vector and the insert DNA was used in 1: 3 ratio for ligation reaction. Ligation mixture was prepared and transformed to E.coli cells. The transformed colonies were selected on LB plate with XIA. The white colonies were screened by colony PCR with respective primers for checking the presence of insert. Restriction digestion was carried out as per the manufacturer?s instruction. The recombinant pBKS plasmids (containing DNA fragment corresponding to domain I&II of Cry2Aa and DNA fragment corresponding to domain III of Cry2Ac) was double digested by KpnI & HindIII and HindIII & XbaI, respectively to release the inserts. The digested product was kept at -20ºC to stop the reaction and analyzed by agarose gel electrophoresis.
Aligning of cloned DNA fragments to construct the chimeric gene, cry2Ax2
The recombinant pBKS containing DNA fragment corresponding to domain I & II of Cry2Aa was double digested by HindIII & XbaI for cloning domain III of Cry2Ac between HindIII & XbaI sites. The linearized recombinant pBKS harboring domain I & II of Cry2Aa and the insert (~441 bp of domain III of Cry2Ac) were excised from 0.8 % agarose gel. The DNA fragments were extracted using gelextraction kit. Recombinant pBluescript vector and insert (DNA fragment of ~441 bp corresponding to domain III of Cry2Ac) were ligated used for transformation of E. coli. The transformants of E. coli colonies were screened by colony PCR and further confirmed by double digestion with KpnI & XbaI restriction enzymes.
Cloning of the chimeric cry2Ax2 gene in expression vector pHT3P2T
The recombinant pBKS plasmid carrying chimeric gene cry2Ax2 was double digested by KpnI and XbaI restriction enzymes to release the cloned DNA fragment. Simultaneously the expression vector pHT3P2T was also double digested using the same enzymes to linearize it. The expression vector pHT3P2T and insert (cry2Ax2) was ligated and transformed into E. coli. The presence of chimeric gene cry2Ax2 was confirmed by restriction digestion of the recombinant pHT3P2T plasmid with the KpnI and XbaI enzymes.
Transformation, screening and analysis of Bt strain 4Q7
Plasmid was isolated from recombinant E.coli colonies and transformed to acrystalliferous Bt strain, 4Q7 through electroporation15. Transformed Bt colonies were selected on LB agar plate containing erythromycin (50 µg/ml). The Bt transformants were screened for the presence of the chimeric gene cry2Ax2 by PCR using 2AFS and 2AR2C primers (specific for DNA fragment corresponding to domain I and II of the chimeric gene cry2Ax2) and 2CF2C and 2ARS (specific for DNA fragment corresponding to domain III of the chimeric gene cry2Ax2). Spore crystal mixture was prepared from recombinant Bt strain, 4Q7 and analysed by SDS-PAGE.
RESULTS
Amplification and cloning of DNA fragment corresponding to domain I & II of Cry2Aa
domain III of Cry2Ac The DNA fragment of about 1465 bp encoding domain I & II of Cry2Aa was amplified from p2Aa with 2AFS and 2AR2C primers by PCR. The ~1465 bp amplicon was subjected for double digestion with KpnI and HindIII. The double digested ~1465 bp amplicon was cloned into the vector pBKS. Recombinant E. coli clones were further screened for the presence of cloned DNA fragment by colony PCR using gene specific primers of the DNA fragment corresponding to domain I & II of Cry2Aa and by restriction digestion. The resultant plasmid is designated as pBKS2AA. The DNA fragment of about 441 bp encoding domain III of Cry2Ac was amplified from p2Ac with 2CF2C and 2ARS primers by PCR. The amplicon (~441 bp) was subjected for double digestion with HindIII and XbaI and cloned into the pBKS vector. Recombinant E. coli clones were further screened for the presence of cloned DNA fragment by colony PCR using gene specific primers of the DNA fragment corresponding to domain III of Cry2Ac and by restriction digestion. The resultant plasmid is designated as pBKS2AC.
Aligning of DNA fragments corresponding to domain I & II of Cry2Aa and domain III of Cry2Ac The recombinant pBKS2AA and pBKS2AC plasmids were double digested with KpnI & HindIII and HindIII & XbaI, respectively. The released insert of ~441 bp from pBKS2AC plasmid was cloned into the linearized recombinant plasmid, pBKS2AA. The recombinant colonies were screened by colony PCR for the presence of gene specific primers of the DNA fragment corresponding to domain I & II of Cry2Aa and domain III of Cry2Ac and by restriction digestion (Fig. 1A). The constructed chimeric gene and its plasmid is named as cry2Ax2 and pBKS2Ax2, respectively. Alignment of nucleotide sequence data obtained from chimeric cry2Ax2 gene with that of cry2Aa1 and cry2Ac1 genes indicated fusion of domain I & II of Cry2Aa and domain III of Cry2Ac in the expected manner.
Cloning of chimeric cry2Ax2 gene in expression vector, pHT3P2T
The recombinant pBKS2Ax2 and expression vector pHT3P2T were double digested with KpnI and XbaI restriction enzymes. The released insert of ~1.9 kb from pBKS2Ax2 plasmid was cloned into the linearized recombinant plasmid, pHT3P2T. The recombinant colonies were confirmed by restriction digestion (Fig. 1B). The recombinant pHT3P2T plasmid harboring cry2Ax2 is named as p2Ax2.
Transformation of Bt strain 4Q7 with p2Ax2
The p2Ax2 plasmids (containing cry2Ax2 gene under the transcriptional control of cry3Aa promoter and cry2Aa terminator) was transformed into the acrystalliferous Bt strain 4Q7 and selected on LB agar plate containing erythromycin (50 µg/ml). Transformants of Bt strain, 4Q7 were screened by PCR using gene specific primers. The amplification of DNA fragments corresponding to domain I & II of Cry2Aa (~1.5 kb) and domain-III of Cry2Ac (~441 bp) was observed in the transformants of Bt strain 4Q7 and there was no amplification in the case of negative control, 4Q7.
Comparison of protein profile of recombinant 4Q7 strains harboring cry2Aa and cry2Ax2 gene expression plasmids
The spore-crystal mixtures obtained from the recombinant Bt strain, 4Q7 harbouring cry2Aa and cry2Ax2 genes under the transcriptional control of cry3Aa promoter and cry2Aa terminator were subjected to SDS-PAGE analysis. The transformants of Bt strain 4Q7 harboring p2Ax2 plasmid did not show any prominent band equivalent to the size of Cry2Aa protein (~65 kDa) where as other positive control, recombinant Bt strain harbouring pHT3P2AT plasmid (Cry2Aa protein), showed the prominent band of ~65 kDa. The protein profile of the recombinant 4Q7 (pHT2Ax2) harboring the chimeric cry2Ax2 gene was similar to that of negative control 4Q7 as shown in fig. 2.
DISCUSSION
A chimeric gene was constructed by replacing the whole DNA fragment of 441 bp encoding domain III of Cry2Ac with that of Cry2Aa. De Maagd et. al.,18 had shown that several Cry1 toxins (e.g. Cry1Ab, Cry1Ac, Cry1Ba, Cry1Ea) with low or no activity against beet armyworm (Spodoptera exigua) become active when their domain III is replaced by that of Cry1Ca. Alternatively, Malvar and Gilmer 19 showed that hybrids of Cry1Ac and Cry1Fa have a wider target spectrum than either of the parental toxins from which they were derived. Rang et al., 20 , demonstrated that domain-III appears to modulate the activity of the chimeric toxins. They also explained that combination of domain -III from Cry1Ab with domain-I and II of Cry1C showed strong activity against insect cell lines than wild type Cry1C. Domain-III of Cry1Ac is involved in the specificity of binding to the putative Cry1Ac receptor from Manduca sexta, aminopeptidase, as well as in the binding to intact membranes 18 . The cloning of DNA fragment of 1465 bp encoding domain I & II of Cry2Aa in pBKS was achieved by domain I & II specific primers by introducing KpnI and HindIII restriction sites at 5? and 3? end respectively. The cloning of DNA fragment of 441 bp encoding domain III of Cry2Ac in pBKS was achieved by its domain specific primers by introducing HindIII and XbaI restriction sites 5? and 3? respectively. HindIII restriction site was created by changing the codons at 485 and 486 positions (CATTTG ? AAGCTT) there by altering the amino acids at 485th position (His ?Lys). The chimeric gene of ~1.9 kb was constructed by aligning domain III of cry2Ac at 5? end between HindIII and XbaI sites of recombinant pBKS containing domain I & II fragment of cry2Aa. The construction of chimeric gene was verified by sequencing and named as cry2Ax2. The cry3Aa gene, isolated from the coleopteran active B. thuringiensis var. tenebrionis19, is a typical example of a non-sporulation-dependent cry gene. It has been shown that the cry3Aa promoter is weak, but significantly expressed during vegetative phase of growth unlike the cry1A promoter, which is sporulation dependent. The expression of cry3Aa is not dependent on sporulation-specific sigma factors either in B. subtilis 21 or in B. thuringiensis 22. The cry3A promoter, although located unusually far upstream of the start codon (position - 558), resembles promoter recognized by the primary sigma factor (σA ) of vegetative cells. Moreover, cry3A expression is increased and prolonged in mutant strains, which are unable to initiate sporulation 23 . In the present study the constructed chimeric gene cry2Ax2 was cloned into expression shuttle vector pHT3PT between cry3Aa promotor and cry2A terminator. In the previous studies the vector has been tested for expression of cry2Aa gene24. The recombinant shuttle vector containing the chimeric gene cry2Ax2 was transferred to Bt strain 4Q7 by electroporation. SDS-PAGE analysis was done for spore crystal mixture harvested from the recombinant clones for Bt strain, 4Q7. Expression of the chimeric gene, cry2Ax2 was not seen as the prominent band of ~65 kDa. Protein profile of the recombinant 4Q7 was same as that in the case of negative control 4Q7. Positive control including recombinant 4Q7 containing cry2Aa gene in the same cassette showed expression as a prominent band of ~65 kDa. The non-expression of the chimeric gene, cry2Ax2, may be attributed to usage of the codons that was introduced to create HindIII restriction site (CATTTG ? AAGCTT) for cloning DNA fragments. The usage for these two codons was compared with the codon usage table for B. thuringiensis from CODON USAGE DATABASE (http://www.kazusa.or.jp/codon/). The usage for CAT codon, which is coding for histidine amino acid was 12.6 and 16.1 per 1000 codons for cry2Aa and cry2Ac genes, respectively. For TTG codon, which is coding for leucine amino acid, the usage was 14.2 and 9.6 per 1000 codons for cry2Aa and cry2Ac genes, respectively. The usage of codons present in HindIII site showed that AAG codon, coding for lysine amino acid, was 1.6 per 1000 codons for both cry2Aa and cry2Ac genes. Another codon, CTT present in the HindIII site, coding leucine amino acid, showed usage of 18.9 and 28.9 per 1000 codons for cry2Aa and cry2Ac genes, respectively. Previous studies on codon usage showed that the frequencies with which individual synonymous codons are used to code their cognate amino acids is quite variable from genome to genome and within genomes, from gene to gene. There is general agreement that codons are translated at different rates25. Hence codon usage has been identified as the single most important factor in prokaryotic gene expression26. In the present study, the introduced codons in the HindIII site of the chimeric cry2Ax2 gene is less preferred for Bt. Therefore, tRNA recognizing the anti-codons in mRNA during translation may not be present in sufficient amount for successful expression of the chimeric gene in the recombinant Bt strain.
CONCLUSION
The expression studies of the newly constructed chimeric cry2Ax2 gene may be done after changing the introduced codons back to the most preferred one (CATTTG ? AAGCTT) by site directed mutagenesis. In previous study, silent mutation of a less preferred codon for an amino acid to more preferred one have increased the expression to four fold27 . Expression of the newly constructed chimeric cry2Ax2 gene may be achieved in the recombinant Bt strain after site directed mutagenesis. The cry2A genes are known to be expressed in recombinant E. coli strains28,29. The codons introduced in the present study are not less preferred by E. coli. Therefore, expression of the newly constructed gene by a suitable promoter in E. coli may also be successful. After getting the chimeric gene expressed, it can be used for further bioassays to assess its toxicity against lepidopteron pest.
Englishhttp://ijcrr.com/abstract.php?article_id=2214http://ijcrr.com/article_html.php?did=22141. Schnepf E, Crickmore N, Van Rie J, Lerecurs D, Baum J, Feitelson J, et al. B. thuringiensis and its pesticidal crystal proteins. Microbiol Mol Biol Rev 1998; 62: 775-806.
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13. Karlova R, Weeman-Hendriks M, Naimov S, Ceron J, Dukiandjiev S and de Maagd RA . Bacillus thuringiensis δ- endotoxin Cry1Ac domain–III enhances activity against Heliothis virescens in some, but not all Cry 1-Cry1Ac hybrids. 2005;J Invertebr Pathol., 88: 169- 172.
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Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-5241210EnglishN-0001November30HealthcareSPECTROPHOTOMETRIC DETERMINATION OF SPARFLOXACIN IN BULK AND DOSAGE FORMS
English2733Okorie HNEnglish Mbah CJEnglishA spectrophotometric study of sparfloxacin is described. The method is based on the charge-transfer complexation between sparfloxacin as n-electron donor with chloranilic acid as -acceptor to form a violet-coloured complex having absorption maximum at 530 nm. Beer?s plot is obeyed in the concentration range of 5-30 g/ml. Results of theanalysis of this method were validated statistically by recovery studies. The proposed
method is simple, accurate and precise for the quantitative determination of sparfloxacin in bulk and tablet formulations.
EnglishSparfloxacin, spectrophotometry, chloranilic acid.INTRODUCTION
Sparfloxacin, 5-amino-1-cyclopropyl-7- (cis-3, 5-dimethyl-1-piperazinyl)-6,8- difluoro-1, 4-dihydro-4-oxo-3- quinolinecarboxylic acid is a difluoroquinolone antibacterial agent belonging to the third generation quinolones. Clinically, it is very effective in the treatment of streptococci infections. Its mechanism of action involves the inhibition of DNA synthesis by promoting cleavage of bacterial DNA in the DNA-enzyme complexes of DNA gyrase and type iv topoisomerase, resulting in rapid bacterial death1 . The drug is not official in any pharmacopoeia, hence no official method is available for the estimation of the drug in formulations. A number of analytical methods used for the determination of sparfloxacin in pure and dosage forms include electrochemistry2-5 ; UV-Visible spectrophotometry6-9 ; HPLC 10-13 . Chloranilic acid, 2,5-dichloro-3,6- dihydroxy-p-benzoquinone has been used largely as a spectrophotometric reagent for the determination of some organic compounds containing lone pair of electrons14-18, but not hitherto in the assay of sparfloxacin. The present work describes the spectrophotometric determination of sparfloxacin in bulk and pharmaceutical formulations using chloranilic acid as a chromogenic reagent.
MATERIALS AND METHODS
Materials: Sparfloxacin (Micro Labs Limited.92, Sipcot, Hosur. India), all other chemicals were of analytical grade. Freshly prepared 0.5 % (w/v) chloranilic acid solution in dioxan. A Hitachi UV/VIS spectrophotometer, model 2000 (Japan) was used for absorbance measurements.
Standard solution: A stock solution of sparfloxacin (50 g/ml) was prepared by dissolving the required amount in methanol. Standard solutions of the analyte (5-30 g/ml) were prepared by serial dilution of the stock solution.
Proposed procedure: An aliquot of the standard solution containing sparfloxacin was transferred into a 10 ml volumetric flask. A 1-ml volume of chloranilic acid solution (500 g/ ml) was added and the contents were mixed thoroughly. After 30 min standing, the volume was made up with dioxan and the absorbance of the solution was measured at 530 nm against reagent blank.
Procedure for assay of dosage forms: Ten tablets of the drug were weighed and ground to a fine powder. An adequate amount of the powder was transferred into a beaker. The powder was dissolved in methanol by stirring for 15 min. The mixture was filtered to a volumetric flask (100 ml) through Whatman filter paper No.41. The filtrate and washings were diluted to volume with methanol. A suitable volume of this solution was treated as described under proposed procedure and the drug content was evaluated. The results are given in Table 2
Procedure for recovery of sparfloxacin: To study the recovery of sparfloxacin, samples were prepared by mixing known amounts of pure sparfloxacin with portions of commercial preparation. The mixtures obtained were assayed by proposed method and the results are presented in Table 3.
RESULTS AND DISCUSSION Spectrophotometric characteristic of the sparfloxacin-chloranilic acid system: A violet-coloured complex with a ratio of sparfloxacin to reagent of 1:1 was formed when chloranilic acid solution was added to sparfloxacin solution. The complex exhibited a max at 530 nm while the reagent showed a max at 434 nm. An absorbance of the complex using an aliquot of the standard solution was measured at 530 nm at 30 min interval over a period of 2h. No change in the initial absorbance was observed indicating that the colour of the complex is fairly stable. Beer?s law was obeyed in the range of 5-30 g/ml. Beer?s law range, molar absorptivity, slope, linear least-square analysis are given in Table 1.
Optimization of reaction conditions: Effect of chloranilic acid concentration: The effect of chloranilic acid concentration on the colour development was studied. It was observed that 1 ml of 0.5 % (w/v) chloranilic acid solution produced maximum colour intensity (Fig. 1).
Effect of reacting time: The colour product developed rapidly after addition of the reagent attaining maximum intensity after 30 min at room temperature (Fig. 2). The colour was stable for over 2 h.
Stoichiometric relationship: In order to establish the composition of the charge-transfer complex, the molar ratio method and Job?s method of continuous variation using equimolar solutions of the drug (0.004 M) and reagent (0.004 M) were studied. In the molar ratio method, the concentration of the drug was kept constant while varying the concentration of the reagent in the series of solutions prepared. In the Job?s method, equimolar solutions of the drug and the reagent were mixed in complimentary proportions to a fixed total volume. The results obtained indicate that the composition of chargetransfer complex was (1:1) drug to reagent (Fig. 3). In conclusion, the proposed spectrophotometric method was applied in the determination of sparfloxacin in bulk and pharmaceutical formulations. The method is simple, accurate, reproducible and the statistical analysis has good agreement with reported methods. The optimum conditions for the proposed method have been established and the method has shown a reasonable tolerance towards excipients. Finally, due to the minimum time required for the complexation to be complete, the proposed method can be employed for the routine analysis of sparfloxacin from bulk and tablet dosage form in quality control laboratories.
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