Astaxanthin protects oxidative stress mediated DNA damage and enhances longevity in Saccharomyces cerevisiae.

Reactive oxygen species (ROS) have long been found to play an important role in oxidative mediated DNA damage. Fortunately, cells possess an antioxidant system that can neutralize ROS. However, oxidative stress occurs when antioxidants are overwhelmed by ROS or impaired antioxidant pathways. This study was carried out to find the protective effect of astaxanthin on the yeast DNA repair-deficient mutant cells under hydrogen peroxide stress. The results showed that astaxanthin enhances the percent cell growth of rad1∆, rad51∆, apn1∆, apn2∆ and ogg1∆ cells. Further, the spot test and colony-forming unit count results confirmed that astaxanthin protects DNA repair mutant cells from oxidative stress. The DNA binding property of astaxanthin studied by in silico and in vitro methods indicated that astaxanthin binds to the DNA in the major and minor groove, and that might protect DNA against oxidative stress induced by Fenton's reagent. The intracellular ROS, 8-OHdG level and the DNA fragmentation as measured by comet tail was reduced by astaxanthin under oxidative stress. Similarly, reduced nuclear fragmentation and chromatin condensation results suggest that astaxanthin might reduce apoptosis. Finally, we show that astaxanthin decreases the accumulation of mutation rate and enhances the longevity of DNA repair-deficient mutants' cells during a chronological lifespan.

Changing trends in the endourological management of urolithiasis in anomalous kidneys.

OBJECTIVES: To present our single-centre experience of urolithiasis management in anomalous kidneys, highlighting the need for an algorithm-based approach in deciding the appropriate treatment method for each type. PATIENTS AND METHODS: We retrospectively reviewed the data of all the patients who presented to our renal unit with urolithiasis in an anomalous kidney between January 1990 and July 2014. The peri-operative characteristics of all the patients, along with management approach, were studied. RESULTS: Percutaneous nephrolithotomy (PCNL) was the most common surgical technique used, followed by flexible ureterorenoscopy (URS). Pain was the most common presentation in these patients. The overall stone clearance rate associated with PCNL and flexible URS was 85.8% in cases of anomalous kidneys in this series. The stone clearance rates in ectopic kidneys, horseshoe kidneys, malrotated kidneys and autosomal dominant polycystic kidneys associated with PCNL and flexible URS were similar: 86%, 88%, 76% and 83%, respectively, and 81.2%, 80%, 92% and 100%, respectively . Based on our results, we developed a stone management algorithm. CONCLUSION: The management of renal calculi in patients with renal anomalies may vary based on the stone size, location, density, pelvicalyceal system anatomy and drainage. An algorithm-based approach could help surgeons decide the appropriate treatment in this population.

Evaluation of in vivo antioxidant potential of Syzygium jambos (L.) Alston and Terminalia citrina Roxb. towards oxidative stress response in Saccharomyces cerevisiae.

Excessive production and restricted elimination of free radicals like superoxide, hydroxyl radical (·OH), anion radical (O2 ·-), and non-radical hydrogen peroxide (H2O2) are related to the development of cancer, arteriosclerosis, arthritis and neurodegenerative diseases. According to a report of World Health Organisation, about 80% of the population living in the developing countries predominantly depends on the traditional medicine for their primary healthcare. Plants possess innate ability to synthesize a wide variety of enzymatic and non-enzymatic antioxidants capable of attenuating ROS-induced oxidative damage. The ethanolic leaf extracts of Syzygium jambos L. and Terminalia citrina Roxb. exhibited a significant in vitro antioxidant activity when compared with natural antioxidant, ascorbic acid. The extracts also provided strong cellular protection against the damaging effects of H2O2 induced oxidative stress in the mutant strains (tsa1Δ and sod1Δ) of Saccharomyces cerevisiae. The GC-MS analysis of the leaf extracts revealed the presence of phytoconstituents majorly constituting of terpenes, vitamin and fatty acids contributing to the antioxidant property. The plant extracts may serve as a potential source of exogenous antioxidants to combat the undesirable effects of oxidative stress.

Determination of antioxidant potential of Acacia nilotica leaf extract in oxidative stress response system of Saccharomyces cerevisiae.

BACKGROUND: From ancient times, plants and plant-derived products have been used as folkloric medicines for a variety of health disorders owing to their tremendous therapeutic potential. The present study aimed to determine the antioxidant efficacy of crude Acacia nilotica extract in the oxidative stress response system of Saccharomyces cerevisiae as a model organism. RESULTS: Acacia nilotica showed significant antioxidant activity, with IC50 values of 75.157 and 159.57 µg mL-1 for 2,2-diphenyl-1-picrylhydrazyl radical and hydroxyl radical scavenging activities respectively at a concentration of 500 µg mL-1 . The total antioxidant activity of A. nilotica showed an ascorbic acid equivalent of 152.79 ± 7.43 µg mL-1 . The presence of phytoconstituents such as phytol and α-tocopherol from gas chromatography/mass spectrometry analysis confirmed the potential of A. nilotica as an antioxidant. The results were validated using the stress response mechanism in S. cerevisiae wild type and its isogenic deletion strains sod1Δ and tsa1Δ. Acacia nilotica substantially neutralized reactive oxygen species generated by hydrogen peroxide in mutant strains, as evident from spot assay and fluorescence assay using fluorescence microscopy and intensity studies. CONCLUSION: The results suggested the efficacy of A. nilotica as a potent antioxidant in the S. cerevisiae system for the first time and its use in neutraceuticals/therapeutics. © 2017 Society of Chemical Industry.

Utility of QuantiFERON®-TB Gold test in diagnosis and management of suspected tubercular uveitis in India.

The tuberculin skin test, used to detect latent systemic tuberculosis (TB), has its limitations. The utility of interferon-gamma assays, found useful in the diagnosis of latent TB, is still unestablished in tubercular uveitis. We present the results of QuantiFERON(®)-TB Gold (QFT-G) test and its relevance in the diagnosis and management of suspected tubercular uveitis in India. All suspected tubercular uveitis patients seen at our uveitis clinic between October 2006 and June 2008 who underwent relevant blood investigations, chest X-rays, Mantoux tests and QFT-G tests were included. Clinical profile, systemic correlation and outcome with treatment were analysed. Fifty suspected tubercular uveitis patients underwent QFT-G testing. The age range of the patients was 6-55 years (mean 32.66 years). Seven patients presented with active and three with a past history of systemic TB. The QFT-G test was positive in 29 patients. Radiological findings of TB were seen in four patients with a positive QFT-G and one patient with a negative QFT-G test. In 11 patients both QFT-G and Mantoux tests were positive. Eighteen Mantoux-negative patients were QFT-G-positive. Significantly, no patient with a positive Mantoux had a negative QFT-G test. Of the 32 patients with posterior uveitis, 17 patients had serpiginous choroiditis, four patients had a choroidal granuloma, six patients had multifocal choroiditis, four patients had retinal vasculitis, and one patient had a subretinal abscess. All QFT-G-positive patients were treated with anti-tuberculosis therapy as well as systemic steroids with a favorable clinical outcome. Our study shows that the QFT-G test is very useful in the diagnosis and management of suspected ocular TB. It was found to be very sensitive in identifying latent TB patients who, upon treatment, had a significantly reduced frequency of recurrences. It was more sensitive than the Mantoux test and is not significantly affected by previous treatment with systemic steroids or immunosuppressives. A negative QFT-G test can also be used as an adjunct before initiation of systemic steroids or immunosuppressives in uveitic patients particularly in an endemic setting like India.

Molecular characterization of three CYP450 genes reveals their role in withanolides formation and defense in Withania somnifera, the Indian Ginseng.

The medicinal properties of Ashwagandha (Withania somnifera) are attributed to triterpenoid steroidal lactones, withanolides, which are proposed to be derived from phytosterol pathway, through the action of cytochrome P450 (CYP450) enzymes. Here, we report the characterization of three transcriptome-mined CYP450 genes (WsCYP749B1, WsCYP76 and WsCYP71B10), which exhibited induced expression in response to methyl jasmonate treatment indicating their role in secondary metabolism. All three WsCYP450s had the highest expression in leaf compared to other tissues. In planta characterization of WsCYP450s through virus induced gene silencing (VIGS) and transient overexpression approaches and subsequent metabolite analysis indicated differential modulation in the accumulation of certain withanolides in W. somnifera leaves. While WsCYP749B1-vigs significantly enhanced withaferin A (~ 450%) and reduced withanolide A (~ 50%), its overexpression drastically led to enhanced withanolide A (> 250%) and withanolide B (> 200%) levels and reduced 12-deoxywithastramonolide (~ 60%). Whereas WsCYP76-vigs led to reduced withanolide A (~ 60%) and its overexpression increased withanolide A (~ 150%) and reduced 12-deoxywithastramonolide (~ 60%). Silencing and overexpression of WsCYP71B10 resulted in significant reduction of withanolide B (~ 50%) and withanolide A (~ 60%), respectively. Further, while VIGS of WsCYP450s negatively affected the expression of pathogenesis-related (PR) genes and compromised tolerance to bacteria P. syringae DC3000, their overexpression in W. somnifera and transgenic tobacco led to improved tolerance to the bacteria. Overall, these results showed that the identified WsCYP450s have a role in one or several steps of withanolides biosynthetic pathway and are involved in conferring tolerance to biotic stress.

Betulinic acid mitigates oxidative stress-mediated apoptosis and enhances longevity in the yeast Saccharomyces cerevisiae model.

Betulinic acid (BA), a pentacyclic triterpenoid found in certain plant species, has been reported to have several health benefits including antioxidant and anti-apoptotic properties. However, the mechanism by which BA confers these properties is currently unknown. Saccharomyces cerevisiae, a budding yeast with a short life cycle and conserved cellular mechanism with high homology to humans, was used as a model for determining the role of BA in aging and programmed cell death (PCD). Treatment with hydrogen peroxide (H2O2) exhibited significantly increased (30-35%) survivability of antioxidant (sod1Δ, sod2Δ, cta1Δ, ctt1Δ, and tsa1Δ) and anti-apoptotic (pep4Δ and fis1Δ) mutant strains when cells were pretreated with BA (30 µM) as demonstrated in spot and CFU (Colony forming units) assays. Measurement of intracellular oxidation level using the ROS-specific dye H2DCF-DA showed that all tested BA-pretreated mutants exhibited decreased ROS than the control when exposed to H2O2. Similarly, when mutant strains were pretreated with BA and then exposed to H2O2, there was reduced lipid peroxidation as revealed by the reduced malondialdehyde content. Furthermore, BA-pretreated mutant cells showed significantly lower apoptotic activity by decreasing DNA/nuclear fragmentation and chromatin condensation under H2O2-induced stress as determined by DAPI and acridine orange/ethidium bromide staining. In addition, BA treatment also extended the life span of antioxidant and anti-apoptotic mutants by ∼10-25% by scavenging ROS and preventing apoptotic cell death. Our overall results suggest that BA extends the chronological life span of mutant strains lacking antioxidant and anti-apoptotic genes by lowering the impact of oxidative stress, ROS levels, and apoptotic activity. These properties of BA could be further explored for its use as a valuable nutraceutical.

Oxidative stress alleviating potential of galactan exopolysaccharide from Weissella confusa KR780676 in yeast model system.

In the present study, galactan exopolysaccharide (EPS) from Weissella confusa KR780676 was evaluated for its potential to alleviate oxidative stress using in vitro assays and in vivo studies in Saccharomyces cerevisiae (wild type) and its antioxidant (sod1∆, sod2∆, tsa1∆, cta2∆ and ctt1∆), anti-apoptotic (pep4∆ and fis1∆) and anti-aging (sod2∆, tsa1∆ and ctt1∆)) isogenic gene deletion mutants. Galactan exhibited strong DPPH and nitric oxide scavenging activity with an IC50 value of 450 and 138 µg/mL respectively. In the yeast mutant model, oxidative stress generated by H2O2 was extensively scavenged by galactan in the medium as confirmed using spot assays followed by fluorescencent DCF-DA staining and microscopic studies. Galactan treatment resulted in reduction in the ROS generated in the yeast mutant cells as demonstrated by decreased fluorescence intensity. Furthermore, galactan exhibited protection against oxidative damage through H2O2 -induced apoptosis inhibition in the yeast mutant strains (pep4∆ and fis1∆) leading to increased survival rate by neutralizing the oxidative stress. In the chronological life span assay, WT cells treated with galactan EPS showed 8% increase in viability whereas sod2∆ mutant showed 10-15% increase indicating pronounced anti-aging effects. Galactan from W. confusa KR780676 has immense potential to be used as a natural antioxidant for nutraceutical, pharmaceutical and food technological applications. As per our knowledge, this is the first report on in-depth assessment of in vivo antioxidant properties of a bacterial EPS in a yeast deletion model system.

Repositioning antispasmodic drug Papaverine for the treatment of chronic myeloid leukemia.

BACKGROUND: Papaverine is a benzylisoquinoline alkaloid from the plant Papaver somniferum (Opium poppy). It is approved as an antispasmodic drug by the US FDA and is also reported to have anti-cancer properties. Here, Papaverine's activity in chronic myeloid leukemia (CML) is explored using Saccharomyces cerevisiae, mammalian cancer cell lines, and in silico studies. METHODS: The sensitivity of wild-type and mutant (anti-oxidant defense, apoptosis) strains of S. cerevisiae to the drug Papaverine was tested by colony formation, spot assays, and AO/EB staining. In vitro cytotoxic effect was investigated on HCT15 (colon), A549 (lung), HeLa (cervical), and K562 (Bcr-Abl positive CML), and RAW 264.7 cell lines; cell cycle, mitochondrial membrane potential, ROS detection analyzed in K562 cells using flow cytometry and apoptotic markers, Bcr-Abl signaling pathways examined by western blotting. Molecular docking and molecular dynamics simulation of Papaverine against the target Bcr-Abl were also carried out. RESULTS: Investigation in S. cerevisiae evidenced Papaverine induces ROS-mediated apoptosis. Subsequent in vitro examination showed that CML cell line K562 was more sensitive to the drug Papaverine. Papaverine induces ROS generation, promotes apoptosis, and inhibits Bcr-Abl downstream signaling. Papaverine acts synergistically with the drug Imatinib. Furthermore, the docking and molecular dynamic simulation studies supported that Papaverine binds to the allosteric site of Bcr-Abl. CONCLUSION: The data presented here have added support to the concept of polypharmacology of existing drugs and natural compounds to interact with more than one target. This study provides a proof-of-concept for repositioning Papaverine as an anti-CML drug.

Phacoemulsification with intraocular lens implantation in juvenile idiopathic arthritis.

BACKGROUND AND OBJECTIVE: To evaluate outcomes of phacoemulsification cataract surgery with intraocular lens (IOL) implantation in children and adults with juvenile idiopathic arthritis-associated uveitis. PATIENTS AND METHODS: Charts of 7 patients (10 eyes) with juvenile idiopathic arthritis-associated uveitis who had phacoemulsification cataract surgery between December 2002 and March 2005 were analyzed. RESULTS: Ten eyes of 7 patients had phacoemulsification with IOL implantation done by a single surgeon. A heparin surface modified IOL was used in 7 eyes and a foldable acrylic IOL was used in 3 eyes. At final follow-up, 70% of eyes had a visual acuity of 20/40 or better and 30% had improved visual acuity to 20/60. Posterior capsular opacification was found in 2 eyes and anterior capsular fibrosis in 1 eye. CONCLUSION: Keys to success following cataract surgery in juvenile idiopathic arthritis are the age of the child, the absence of amblyopia, and strict preoperative and postoperative control of inflammation.

Coats-like response in tubercular subretinal abscess masquerading as melanoma as the initial manifestation of miliary tuberculosis.

Ocular neoplasia can masquerade as an inflammatory condition and vice-versa, which if untreated, can lead to loss of vision or even life. We present a young immunocompetent adult male referred to us as choroidal melanoma. He had a large atypical choroidal mass and exudative retinal detachment. He had no systemic complaints and ocular lesions were the first manifestation of disseminated disease. Aqueous polymerase chain reaction for Mycobacterium tuberculosis (MTB) was negative, but Mantoux, chest radiology, acid-fast bacilli, and caseation necrosis on pleural nodule histopathology confirmed tuberculosis (TB). While on antitubercular therapy, a rare "Coats'-like response" was noted which eventually resolved completely with treatment.

Polymerase chain reaction-proven tuberculous anterior segment mass mimicking juvenile xanthogranuloma in a child.

Mass lesions arising from the anterior segment in children involving the iris and ciliary body can be of myogenic, neurogenic, or hematogenic/vascular origin. These include nevi, melanomas, adenoma, adenocarcinoma, cysts, metastatic tumours among others. Multiple iris mass lesions due to tuberculosis in children are rare. We present an uncommon atypical presentation of multiple anterior segment mass lesions referred to us as neoplasia. Although excision biopsy can be diagnostic, it was deferred and anterior chamber tap was done. Aqueous cytology was suspicious of juvenile xanthogranuloma (JXG) but polymerase chain reaction (PCR) confirmed tuberculous etiology. Treatment with antituberculous therapy (ATT) and steroids lead to complete resolution of the lesions.

Tubercular panophthalmitis in a patient with human immunodeficiency virus infection: Proven clinicopathologically and by molecular diagnostic tests.

Ocular tuberculosis (OTB) in patients with human immunodeficiency virus (HIV) commonly presents as choroidal tubercles or granuloma. We report a rare presentation of OTB with hypopyon granulomatous uveitis in a patient with HIV on highly active antiretroviral therapy (HAART). Aqueous humor polymerase chain reaction (PCR) was positive for Mycobacterium tuberculosis (MTB). Antitubercular therapy (ATT) was initiated despite which it progressed to scleral abscess and panophthalmitis. Enucleation with ball implantation was done. Histopathology revealed caseating granuloma with numerous acid-fast bacilli (AFB) and real-time PCR showed increased copies of MTB genome. After a full course of ATT, he had stable ocular and systemic condition at final follow-up.

Tubercular subretinal abscess in a pediatric intermediate uveitis patient on methotrexate.

Pediatric intermediate uveitis (IU), usually idiopathic, can also be associated with tuberculosis (TB) and sarcoidosis. A 14-year-old girl was diagnosed with idiopathic IU after ruling out TB and sarcoid. She was treated with oral steroids and methotrexate (MTX) with good inflammation control. One year later, she presented with subretinal (SR) abscess. Lab tests were still negative but aqueous polymerase chain reaction confirmed TB. With antituberculosis treatment, complete resolution of the lesion was noted. The likelihood of a change in phenotype of ocular TB, from an IU to TB SR abscess or a possible reactivation of latent TB due to MTX are discussed.

Astaxanthin reduces perfluorooctanoic acid cytotoxicity in Saccharomyces cerevisiae.

Perfluorooctanoic acid (PFA) has been identified as an environmental contaminant of high concern for human health. In this study, we demonstrated that PFA induces a dose (0 to 1.5 mM) dependent cytotoxicity in S. cerevisiae cells which can be rescued by astaxanthin. The percent sensitivity induced by PFA and the cell protection offered by astaxanthin (30 µM) were demonstrated by CFU counts and spots. The increase in intracellular ROS, superoxide dismutase (SOD), glutathione and lipid peroxidation levels in PFA treated cells suggested that increased oxidative stress resulted in yeast cell death. In contrast, decreased ROS level, increased SOD activity, reduced glutathione and decreased lipid peroxidation by astaxanthin supplementation suggest that the cells are protected from the PFA induced oxidative stress mediated cytotoxicity. Reduced chromatin condensation and nuclear fragmentation in astaxanthin pre-treated cells indicate that astaxanthin rescued the cells from PFA induced apoptosis. Our overall results suggest that PFA induces oxidative stress-mediated cytotoxicity in yeast cells, which were rescued by astaxanthin treatment.

Astaxanthin supplementation reduces dichlorvos-induced cytotoxicity in Saccharomyces cerevisiae.

This study evaluates the protective effect of astaxanthin against dichlorvos cytotoxicity in yeast Saccharomyces cerevisiae. Dichlorvos induce a dose-dependent cytotoxicity in yeast cells, which is mediated by oxidative stress. Our experimental results showed pre-treatment with astaxanthin enhances cell viability by 20-30% in yeast cells exposed to dichlorvos. A decrease in DCF fluorescence intensity and lipid peroxidation, increased SOD activity, and glutathione levels in astaxanthin-treated cells indicate that astaxanthin protected the cells against dichlorvos-induced oxidative stress. Reduced chromatin condensation and nuclear fragmentation in astaxanthin pre-treated cells also indicate that astaxanthin rescued the cells from dichlorvos-induced apoptosis. Our overall results suggest that dichlorvos induces oxidative stress-mediated cytotoxicity in yeast cells, and that was rescued by astaxanthin pre-treatment.

Familial Blau syndrome:First molecularly confirmed report from India.

Blau syndrome (BS) is a rare autoinflammatory disorder characterized by the clinical triad of arthritis, uveitis, and dermatitis due to heterozygous gain-of-function mutations in the NOD2 gene. BS can mimic juvenile idiopathic arthritis (JIA)-associated uveitis, rheumatoid arthritis, and ocular tuberculosis. We report a family comprising a mother and her two children, all presenting with uveitis and arthritis. A NOD2 mutation was confirmed in all the three patients - the first such molecularly proven case report of familial BS from India.

Introduction and immunopathogenesis of acquired immune deficiency syndrome.

India has a large number of patients with acquired immune deficiency syndrome (AIDS), the third largest population of this group in the world. This disease was first described in patients with Pneumocystis pneumonia in 1981. Ocular lesions can occur at any stage of the disease but are more commonly seen at the late stages. Human immunodeficiency virus (HIV), the causative agent of AIDS is a retrovirus with RNA genome and a unique 'Reverse transcriptase enzyme' and is of two types, HIV-1 and 2. Most human diseases are caused by HIV-1. The HIV-1 subtypes prevalent in India are A, B and C. They act predominantly by reducing the CD4+ cells and thus the patient becomes susceptible to opportunistic infections. High viral titers in the peripheral blood during primary infection lead to decrease in the number of CD4+ T lymphocytes. Onset of HIV-1-specific cellular immune response with synthesis of HIV-1 specific antibodies leads to the decline of plasma viral load and chronification of HIV-1 infection. However, the asymptomatic stage of infection may lead to persistent viral replication and a rapid turnover of plasma virions which is the clinical latency. During this period, there is further decrease in the CD4+ counts which makes the patient's immune system incapable of controlling opportunistic pathogens and thus life-threatening AIDS-defining diseases emerge. Advent of highly active antiretroviral treatment (HAART) has revolutionized the management of AIDS though there is associated increased development of immune recovery uveitis in a few of these patients.

Anterior segment manifestations of human immunodeficiency virus/acquired immune deficiency syndrome.

Ocular complications are known to occur as a result of human immunodeficiency virus (HIV) disease. They can be severe leading to ocular morbidity and visual handicap. Cytomegalovirus (CMV) retinitis is the commonest ocular opportunistic infection seen in acquired immune deficiency syndrome (AIDS). Though posterior segment lesions can be more vision-threatening, there are varied anterior segment manifestations which can also lead to ocular morbidity and more so can affect the quality of life of a HIV-positive person. Effective antiretroviral therapy and improved prophylaxis and treatment of opportunistic infections have led to an increase in the survival of an individual afflicted with AIDS. This in turn has led to an increase in the prevalence of anterior segment and adnexal disorders. Common lesions include relatively benign conditions such as blepharitis and dry eye, to infections such as herpes zoster ophthalmicus and molluscum contagiosum and malignancies such as squamous cell carcinoma and Kaposi's sarcoma. With the advent of highly active antiretroviral therapy, a new phenomenon known as immune recovery uveitis which presents with increased inflammation, has been noted to be on the rise. Several drugs used in the management of AIDS such as nevirapine or indinavir can themselves lead to severe inflammation in the anterior segment and adnexa of the eye. This article is a comprehensive update of the important anterior segment and adnexal manifestations in HIV-positive patients with special reference to their prevalence in the Indian population.

Expression, purification, and characterization of pyruvate kinase from Mycobacterium tuberculosis: A key allosteric regulatory enzyme.

Background: The current research aims to isolate pyruvate kinase (Pyk) gene from Mycobacterium tuberculosis and expression of the gene (Rv1617) to obtain a purified enzyme. The enzyme activity and secondary structural features were assessed through biochemical assays and circular dichroism (CD) spectroscopy, respectively. Methods: The Pyk-encoding gene from the complete genome of M. tuberculosis was cloned, sequenced, and expressed in Escherichia coli BL21 (DE3). The enzyme was purified by nickel-nitrilotriacetic acid affinity chromatography and enzyme activity was determined by a lactose dehydrogenase-coupled assay system. Further, far ultraviolet CD spectra of the enzyme and the substrate bound enzyme were analyzed using a Jasco J712 spectrophotometer. Results: A single protein with an approximate molecular mass of 54 kDa was purified and a specific activity of 5.31 units/mg was determined from purified M. tuberculosis Pyk. The activity of the enzyme indicating a protein is defined by separate domain for each catalytic function. The secondary structure analysis of CD spectra of the recombinant Pyk has revealed a content of 17% α-helix, 34% ß-sheet, and 49% turns in the enzyme. Conclusion: The growing evidence has impacted M. tuberculosis central carbon metabolism as a key determinant of the survival and pathogenicity in the host. The purified Pyk was observed to have increased enzyme activity in all steps of purification. Retention of Pyk activity indicates a possible catalytic role for the lower part of the glycolytic pathway. The overall results of the spectra obtained from the CD suggest that the substrate phosphoenolpyruvate and adenosine diphosphate binding to the enzyme can cause conformational changes resulting in the exposure or shielding of residues susceptible to modification.