Potukuchi Venkata Gurunadha Krishna Sarma

Angiotensin converting enzyme gene polymorphism in type II diabetics with nephropathy

Nephropathy is an important and a frequent complication of long-term type II diabetic nephropathy. Strong evidence exists that genetic predisposition plays a major role in the development of diabetic nephropathy. Recent studies have implicated association between angiotensin converting enzyme (ACE) insertion/deletion (I/D) gene polymorphism and nephropathy. The deletion gene polymorphism of ACE gene has been shown to be associated with increased activity of this enzyme. This study examines the association of ACE I/D polymorphism with type II diabetes without nephropathy in 30 patients and type II diabetes with nephropathy in 30 patients. The results of the study suggest the association between the DD polymorphism and type II diabetes with nephropathy.

In vitro differentiation of cultured human CD34+ cells into astrocytes

BACKGROUND Astrocytes are abundantly present as glial cells in the brain and play an important role in the regenerative processes. The possible role of stem cell derived astrocytes in the spinal cord injuries is possible related to their influence at the synaptic junctions. AIM The present study is focused on in vitro differentiation of cultured human CD34+ cells into astrocytes. MATERIALS AND METHODS Granulocyte-colony stimulating factor mobilized human CD34+ cells were isolated from peripheral blood using apheresis method from a donor. These cells were further purified by fluorescence-activated cell sorting and cultured in Dulbeccos modified eagles medium. Thus, cultured cells were induced with astrocyte defined medium (ADM) and in the differentiated astrocytes serine/threonine protein kinases (STPK) and glutamine synthetase (GLUL) activities were estimated. The expression of glial fibrillary acidic protein (GFAP) and GLUL were confirmed by reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS The cultured human CD34+ cells differentiated into astrocytes after 11 h of incubation in ADM. The RT-PCR experiment showed the expression of GLUL (1.5 kb) and GFAP (2.9 kb) in differentiated astrocytes. The high enzyme activities of GLUL and STPK in differentiated astrocytes compared with cultured human CD34+ cells confirmed astrocyte formation. CONCLUSION In the present study, in vitro differentiation of stem cells with retinoic acid induction may result in the formation of astrocytes.

Expression of an epitopic region of AspfI, an allergen/antigen/cytotoxin of Aspergillus fumigatus.

The gene for an 18 kD allergen/cytotoxin of Aspergillus fumigatus was cloned in pUC-19 vector and expressed in Escherichia coli JM109. Digestion of this gene with AluI resulted in four fragments of 216bp, 120bp, 39bp and 21bp. These fragments were cloned in the Sma-I site of pUC-19. The recombinants thus, generated after transformation in E. coli JM109, were screened using monoclonal antibodies raised against the AspfI. The fusion protein containing 120 bp AluI fragment was recognised by the MoAb indicating presence of epitope(s) in the 120 bp region. The study indicates a viable strategy for identification and expression of an immunologically active domain of a major allergen/antigen of A. fumigatus for the first time.

Factor V Leiden mutation is not a predisposing factor for acute coronary syndromes.

BACKGROUND The prevalence of Coronary artery disease (CAD) in India has increased considerably over the past few years and could become the number one killer disease if interventions are not done. Factor V Leiden (FVL) mutation and FII G20210A polymorphism are two recently described genetic factors with a propensity towards venous thrombosis. This warrants the investigations for thrombophilia in myocardial infarction patients in India. METHODS The study cohort consisted of 51 patients aged below 50 years presenting with acute coronary syndromes. In both patient group and normal individuals the major risk factors Protein C deficiency, Protein S deficiency, anticardiolipin antibodies, Fibrinogen and Lipoprotein [a] were studied. Factor V Leiden (FVL) G1691A mutation in both control and patient group was looked by using Polymerase chain reaction (PCR) followed by sequencing of the PCR products. RESULTS Our results indicated significantly higher levels of anticardiolipin antibodies and fibrinogen in the patients and absence of FVL (G1691A) mutation in our study cohort. One of the patients (H5) showed insertion of an extra A nucleotide in exon 10 of the Factor V gene resulting in frame shift mutation in this patient. CONCLUSION The results of present study showed absence of FVL mutation in our population. However, there is a need to confirm the above findings on patients from different populations from different parts of the country. The insertion of an extra A in exon 10 in the patient needs to be ascertained to confirm that it is one of its kinds or is prevalent in the population.

Mutations in exons 10 and 11 of human glucokinase result in conformational variations in the active site of the structure contributing to poor substrate binding – explains hyperglycemia in type 2 diabetic patients

Mutations in the glucokinase (GK) gene play a critical role in the establishment of type 2 diabetes. In our earlier study, R308K mutation in GK in a clinically proven type 2 diabetic patient showed, structural and functional variations that contributed immensely to the hyperglycemic condition. In the extension of this work, a cohort of 30 patients with established type 2 diabetic condition were chosen and the exons 10 and 11 of GK were PCR-amplified and sequenced. The sequence alignment showed A379S, D400Y, E300A, E395A, E395G, H380N, I348N, L301M, M298I, M381G, M402R, R308K, R394P, R397S, and S398R mutations in 12 different patients. The structural analysis of these mutated GKs, showed a variable number of β-α-β units, hairpins, β-bulges, strands, helices, helix–helix interactions, β-turns, and γ-turns along with the RMSD variations when compared to wild-type GK. Molecular modeling studies revealed that the substrate showed variable binding orientations and could not fit into the active site of these mutated structures; moreover, it was expelled out of the conformations. Therefore, these structural variations in GK due to mutations could be one of the strongest reasons for the hyperglycemic levels in these type 2 diabetic patients.

Mutations in exons 3 and 7 resulting in truncated expression of human ATP6V1B1 gene showing structural variations contributing to poor substrate binding-causative reason for distal renal tubular acidosis with sensorineural deafness

Distal renal tubular acidosis (dRTA) is an autosomal recessive syndrome results defect in either proximal tubule bicarbonate reabsorption or in distal tubule H+ secretion and is characterized by severe hyperchloraemic metabolic acidosis in childhood. dRTA is associated with functional variations in the ATP6V1B1 gene encoding β1 subunit of H+-ATPase, key membrane transporters for net acid excretion of α-intercalated cells of medullary collecting ducts. In the present study, a 13-year-old male patient suffering with nephropathy and sensorineural deafness was reported in the Department of Nephrology. We predicted improper functioning of ATP6V1B1 gene could be the reason for diseased condition. Therefore, exons 3, 4, and 7 contributing active site of ATP6V1B1 gene was amplified and sequenced (Accession numbers: KF571726, KM222653). The obtained sequences were BLAST searched against the wild type ATP6V1B1 gene which showed novel mutations c.307 A > G, c.308 C > A, c.310 C > G, c.704 T > C, c.705 G > T, c.709 A > G, c.710 A > G, c.714 G > A, c.716 C > A, c.717delC, c.722 C > G, c.728insG, c.741insT, c.753G > C. These mutations resulted in the expression of truncated protein terminating at Lys 209. The mutated ATP6V1B1structure superimposed with wild type showed extensive variations with RMSD 1.336 Å and could not bind to substrate ADP leading to non-functional ATPase. These results conclusively explain these mutations in ATP6V1B1 gene resulted in structural changes causing accumulation of H+ ions contributing to dRTA with sensorineural deafness.

Structural Variations of Human Glucokinase Glu256Lys in MODY2 Condition Using Molecular Dynamics Study

Glucokinase (GK) is the predominant hexokinase that acts as glucose sensor and catalyses the formation of Glucose-6-phosphate. The mutations in GK gene influence the affinity for glucose and lead to altered glucose levels in blood causing maturity onset diabetes of the young type 2 (MODY2) condition, which is one of the prominent reasons of type 2 diabetic condition. In view of the importance of mutated GK resulting in hyperglycemic condition, in the present study, molecular dynamics simulations were carried out in intact and 256 E-K mutated GK structures and their energy values and conformational variations were correlated. Energy variations were observed in mutated GK (3500 Kcal/mol) structure with respect to intact GK (5000 Kcal/mol), and it showed increased γ-turns, decreased β-turns, and more helix-helix interactions that affected substrate binding region where its volume increased from 1089.152 Å2 to 1246.353 Å2. Molecular docking study revealed variation in docking scores (intact = −12.199 and mutated = −8.383) and binding mode of glucose in the active site of mutated GK where the involvement of A53, S54, K56, K256, D262 and Q286 has resulted in poor glucose binding which probably explains the loss of catalytic activity and the consequent prevailing of high glucose levels in MODY2 condition.

Cloning, expression and characterization of glucokinase gene involved in the glucose-6- phosphate formation in Staphylococcus aureus

Glucose-6-phosphate (G-6-P) formation in Staphylococcus aureus is catalysed by glucokinase (glkA) gene under high glucose concentration leading to upregulation of various pathogenic factors; therefore the present study is aimed in the cloning and characterization of glk A gene from S. aureus ATCC12600. The glk A gene was cloned in the Sma I site of pQE 30, sequenced (Accession number: JN645812) and expressed in E. coli DH5α. The recombinant glk A expressed from the resultant glk A 1 clone was purified using nickel metal chelate chromatography, the pure enzyme gave single band in SDS-PAGE with molecular weight of 33kDa. The rglk A showed very high affinity to glucose Km 5.1±0.06mM with Hill coefficient of 1.66±0.032mM. Analysis of glucokinase sequence of S. aureus showed presence of typical ATP binding site and ROK motif CNCGRSGCIE. Sequentially and phylogenetically S. aureus glk A exhibited low identity with other bacterial glk A and 21% homology with human glucokinase (GCK). Functionally, S. aureus glk A showed higher rate of G-6-P formation compared to human GCK which may have profound role in the pathogenesis.

Identification of novel mutations in CD2BP1 gene in clinically proven rheumatoid arthritis patients of south India

Pyogenic Arthritis, Pyoderma gangrenosum, and Acne (PAPA syndrome) is a rare autosomal dominant, auto-inflammatory disease that affects joints and skin. The disease results due to mutations in the cluster of differentiation 2 binding protein 1 (CD2BP1) gene on chromosome 15q24.3. Rheumatoid arthritis (RA) is a common, genetically complex disease that affects the joints with occasional skin manifestations. Studies related to the pathophysiology of inflammation in these two disorders show a certain degree of overlap at genetic level. The present study was done to confirm the existence of such a genetic overlap between PAPA syndrome and RA in south Indian population. In the present study 100 patients who were clinically diagnosed rheumatoid arthritis and 100 apparently healthy controls were chosen and the 15 exons of CD2BP1 gene were PCR-amplified and sequenced. The sequence analysis showed that in exon 3 thirty eight patients revealed presence of novel heterozygous missense mutations p.Glu51Asp, p.Leu57Arg and p.Ala64Thr. In exons 6, 10 and 14 eight patients showed 44 novel missense mutations and two patients showed novel frame shift mutations p.(Met123_Leu416delinsThr) and p.(Thr337Profs*52) leading to truncated protein formation. Such mutations were not seen in controls. Further, the in silico analysis revealed the mutant CD2BP1 structure showed deletion of Cdc15 and SH3 domains when superimposed with the wild type CD2BP1 structure with variable RMSD values. Therefore, these structural variations in CD2BP1 gene due to the mutations could be one of the strongest reasons to demonstrate the involvement of these gene variations in the patients with rheumatoid arthritis.

Challenges in prevention, diagnosis and therapy of emerging fungal diseases. Aspergillosis: A case study

Diseases caused by pathogenic filamentous fungi, are an emerging threat to public health in the wake of increasing incidence of HIV and tuberculosis. At this point, discovery and development of fungal therapeutics and diagnostics are serious challenges for biomedical researchers. Recent technological advances in genomics and proteomics offer great scope for development of preventive and therapeutic measures for fungal diseases.Aspergillus, one of the medically important filamentous pathogenic fungi causes a wide spectrum of clinical disorders ranging from allergic aspergillosis to systemic invasive aspergillosis. Increase in incidence of drug resistance and the cytotoxic effects are two serious limitations of the antifungal drugs presently in use. This is primarily due to lack of understanding of biological mechanisms operative in these fungi. Today, it is possible to understand the biological mechanisms of the fungus for its colonisation, survival and invasion of the host. Future developments based on such leads can result in development of precise and specific diagnostic, therapeutic and preventive measures for a wide clinical spectrum of fungal diseases.