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TITLE
CERTIFICATE
CERTIFICATE
DECLARATION
ACKNOWLEDGEMENT
ABBREVIATIONS
CONTENTS
PREFACE
I. INTRODUCTION
Table 1.1Commonly used anticancer drugs
Table 1.2List of common combinations of drugs
L-Asparaginase as an antitumor drug
Tumor markers
II. SOURCES OF L-ASPARAGINASE
II. A. Screening of Micro organisms for L-asparaginase
MATERIALS AND METHODS
RESULTS AND DISCUSSION
II. B. Optimization of Cultural Conditions for L-asparaginase Production by Aeromonas
MATERIALS AND METHODS
Bacterial strain
Identification of the Bacterial strain
Maintenance of Aeromonas
RESULTS AND DISCUSSION
Fig. II -2. Effect of pH on growth and enzyme production in Aeromonas.
Fig. II -3. Effect of Temperature on growth and enzyme production in Aeromonas.
Fig. II -4. Optimum concentration of Sodium Chloride for growth and enzyme production in Aerouonas.
Table II. 4Effect of salts on production of L-asparaginase in Aeromonas
Fig. II -5. Optimum concentration of I.- Asparagine for growth and enzyme production in Aeromonas.
Fig. II -6. Optimum concentration of L- Glutamine for growth and enzyme production in Aeromonas.
Fig. II -7. Optimum concentration of L- Aspartic acid for growth and enzyme production in Aeromnas.
Fig. II -8. Optimum concentration of Glutamic acid for growth and enzyme production in Aeromonas
Fig. II -9. Optimum concentration of Lactate for growth and enzyme production in Aeroeonas
Pigure: II -10. Optimum concentration of Yeast Extract for growth and enzyme production in Aeromonas.
Table II. 5 Effect of carbon sources on growth and enzyme production in Aeromonas
Table II. 6 Effect of nitrogen sources on growth and enzyme production in Aeromonas
Table II. 7 Effect Of amino acids on growth and enzyme production in Aeromonas
Table II. 8 L-asparaginase production by Aeromonas in synthetic media
III. ISOLATION, PURIFICATION AND CHARACTERIZATION OF ESTUARINE AEROMONAS L-ASPARAGINASE
III. A. Isolation and Purification of Aeromonas L-asparaginase
Medium for growth of Aeromonas
Cultivation of the organism
Harvesting of the cells
DEAE cellulose chromatography
Fig. III -1. DEAE cellulose column Chromatogram.
Concentration and dialysis
Gel filtration
Fig. III -2. Sephadex G-200 co--mn Chromatogram.
Hydroxyapatite chromatography
Fig. III-3. Hydroxyapatite column Chromatogram.
Table 111.1Purification of Aeromonas L-asparaginase
III. B. Characterization of Aeromonas L-asparaginase
Substrate specificity
Effect of substrate concentration
Fig. III -4. Effect of Substrate concentration (lineveaver-Burk Plot)
Effect of enzyme concentration
Fig. III -5. Effect of Enzyme concentration.
Fig. III -6. Effect of pH on Aeromonas L-asparaginase activity.
Effect of pH
Effect of temperature
Fig. III -7. Effect of Temperature on Aeromonas L-asparaginase activity.
pH stability of the enzyme
Heat stability of the enzyme
Fig. III -8. pH stability of Aeromonas L-asparaginase.
Fig. III -9. Heat stability of Aeromonas L-asparaqinase.
Effect of Keto acids
Effect of amino acids
Effect of inorganic ions
Effect of serum and serum constituents
Effect of sulphydryl agents and sulphydryl inhibitors
Effect of dissociating agents
IV. ANTITUMOR PROPERTY OF L-ASPARAGINASE ISOLATED FROM THE AEROMONAS
IV. A. Solid Tumor Reduction Studies
MATERIALS AND METHODS
Experimental animals
Tumor cell lines and their maintenance
Asparaginase preparations
(a) Aeromonas L-asparaginase
(b) E.coli L-asparaginase
Determination of antineoplastic activity of L-asparaginase
Enzyme therapy
RESULTS AND DISCUSSION
Table IV.2 Comparison of antineoplastic activity of Leunase and Aeromonas L-asparaginase
Fig. IV.1 (a) Front view of the tumor growth.
Fig. IV.1 (b) Side view of the tumor growth.
Fig. IV.2 (a) Mouse treated with Aeromonas L-asparaginase.
Fig. IV-2 (b) Mouse treated with Leunase.
Table IV.3 Effect of L-asparaginase isolated from Aeromonas on Ehrlich ascites tumor bearing mice
Table IV.4 Effect of E.coli and Aeromonas L-asparaginases on the growth of Ehrlich ascites tumor in mice
Fig. IV.3. A normal mouse without tumor.
Fig. IV.4. Mouse bearing Ehrlich ascites tumor.
Fig. IV.5 (a) Mouse treated with Aeromonas L-asparaginase.
Fig. IV.5 (b) Mouse treated with Leunase.
IV.B. Biochemical Studies
MATERIALS AND METHODS
I. Carbohydrate Metabolism
II. Lipid Metabolism
1. Extraction of serum and tissues for lipid estimation
2. Estimation of cholesterol
3. Estimation of triglycerides
4. Estimation of phospholipids
III. Protein Metabolism
1. Estimation of protein
2. Estimation of blood urea
IV. Glycoprotein Metabolism
1. Extraction of glycoproteins from the tissues
a) Preparation of dry defatted tissue
b) Papain digestion
2 Estimation of total hexose
3. Estimation of fucose
4. Estimation of sialic acid
V. Assay of Serum Enzymes
1. Estimation of serum lactate dehydrogenase
2. Estimation of serum transaminases
3. Serum phosphatases
(i) Alkaline phosphatase
(ii) Total acid phosphatase
(iii) Tartarate labile acid phosphatase
RESULTS AND DISCUSSION
I. Gain in Weight
II. Carbohydrate Metabolism
Glucose levels in blood
III. Lipid Metabolism
1) Cholesterol
2) Triglycerides
3) Phospholipids
IV. Protein Metabolism
1) Total protein
2) Blood urea
V. Glycoprotein Metabolism
1) Protein bound hexose
2) protein bound fucose
3) Protein bound sialic acid
VI. Serum Enzymes as Tumor Markers
1) Serum lactate dehydrogenase
2) Serum transaminases
3) Serum phosphatases
V. SUMMARY
REFERENCES