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D K Srivastava

Publications and source records attributed to D K Srivastava.

At least 19 recordsLinked to original sources
PubMed2006

Purification of recombinant annexins without the use of phospholipids.

Due to their involvement in a variety of physiological and pathological processes, different isoforms of annexins are being utilized as markers of some human diseases and bio-imaging of tissue injury (due to apoptosis), and have been proposed as drug delivery vehicles. These, in addition to extensive biophysical studies on the role of annexins in organizing lipid domains in biological membranes, have necessitated development of an efficient protocol for producing annexins in bulk quantities. In this paper, we report a one-step purification protocol for annexin a5 without using lipid vesicles or involving any column chromatographic step. Depending on the growth and expression condition, a fraction of recombinant annexin a5 (cloned in pET3d vector) was sequestered into inclusion bodies. When these inclusion bodies were dissolved in 6 M urea, subjected to a 10-fold snap dilution in the presence of 5 mM Ca(2+) and stored overnight at 4 degrees C, annexin a5 was precipitated as a homogenous protein as judged by SDS-PAGE. This one-step purification protocol produced about 35 mg of highly purified annexin a5 per liter of bacterial culture. The annexin a5 purified from inclusion bodies exhibited similar properties to that obtained from the soluble fraction using the conventional lipid-partitioning approach. Our purification protocol for annexin a5 elaborated herein is equally effective for purification of annexin A2, and we believe, will serve as general protocol for purifying other annexins in bulk quantities for diagnostic as well as detailed biophysical studies.

Animals↗
PubMed2006

Formulation of photocleavable liposomes and the mechanism of their content release.

In pursuit of designing photocleavable liposomes as drug delivery vehicles, we synthesized several amphiphilic lipids by connecting stearyl amine (as the non-polar tail) and charged amino acids (as polar heads) via the o-nitrobenzyl derivatives. The lipids containing Glu, Asp, and Lys amino acids were subjected to photocleavage reaction by UV light, and the overall spectral changes of the chromophoric o-nitrobenzyl conjugates were determined as a function of time. The experimental data revealed that the feasibility of the cleavage reaction, nature and magnitude of the spectral changes during the course of the cleavage reaction, and their overall kinetic profiles were dictated by the type of amino acid constituting the polar head groups. The cleavage reactions of the Asp and Glu containing lipids were found to be more facile than that of the lysine-containing lipid. Using these lipids, we formulated photocleavable liposomes, and investigated the photo-triggered release of an encapsulated (within the liposomal lumen) dye as a function of time. The kinetic data revealed that the release of the liposomal content conformed to a two-step mechanism, of which the first (fast) step involved the photocleavage of lipids followed by the slow release of the liposomal content during the second step. The overall mechanistic features intrinsic to the photocleavage of Asp, Glu and Lys containing o-nitrobenzyl conjugated lipids, and their potential applications in formulating liposomes (whose contents can be "unloaded" by the UV light) as drug delivery vehicles are discussed.

Amines↗
PubMed2006

Ultrahigh resolution crystal structures of human carbonic anhydrases I and II complexed with "two-prong" inhibitors reveal the molecular basis of high affinity.

The atomic-resolution crystal structures of human carbonic anhydrases I and II complexed with "two-prong" inhibitors are reported. Each inhibitor contains a benzenesulfonamide prong and a cupric iminodiacetate (IDA-Cu(2+)) prong separated by linkers of different lengths and compositions. The ionized NH(-) group of each benzenesulfonamide coordinates to the active site Zn(2+) ion; the IDA-Cu(2+) prong of the tightest-binding inhibitor, BR30, binds to H64 of CAII and H200 of CAI. This work provides the first evidence verifying the structural basis of nanomolar affinity measured for two-prong inhibitors targeting the carbonic anhydrases.

Carbonic Anhydrase I↗
PubMed2005

Molecular basis for the origin of differential spectral and binding profiles of dansylamide with human carbonic anhydrase I and II.

Sulfonamide derivatives serve as potent inhibitors of carbonic anhydrases (CAs), and a few such inhibitors have been currently used as drugs for the treatment of different pathogenic conditions in humans. In pursuit of designing the isozyme-specific inhibitors of human CAs, we observed that the fluorescence spectral properties and binding profiles of a fluorogenic sulfonamide derivative, 5-(dimethylamino)-1-naphthalenesulfonamide (dansylamide, DNSA), were markedly different between the recombinant forms of human carbonic anhydrase I (hCA I) and II (hCA II). The kinetic evaluation of the overall microscopic pathways for the binding of DNSA to hCA I versus hCA II revealed that the protein isomerization step served as a major determinant of the above discrepancy. Arguments are presented that the detailed structural-functional investigations of enzyme-ligand interactions may provide insights into designing the isozyme-specific inhibitors of CAs.

Binding Sites↗
PubMed2005

Spacer-based selectivity in the binding of "two-prong" ligands to recombinant human carbonic anhydrase I.

Benzenesulfonamide and iminodiacetate (IDA)-conjugated Cu(2+) independently interact at the active site and a peripheral site of carbonic anhydrases, respectively [Banerjee, A. L., Swanson, M., Roy, B. C., Jia, X., Haldar, M. K., Mallik, S., and Srivastava, D. K. (2004) J. Am. Chem. Soc. 126, 10875-10883]. By attaching IDA-bound Cu(2+) to benzenesulfonamide via different chain length spacers, we synthesized two "two-prong" ligands, L1 and L2, in which the distances between Cu(2+) and NH(2) group of sulfonamide were 29 and 22 A, respectively. We compared the binding affinities of L1 and L2, vis-a-vis their parent compound, benzenesulfonamide, for recombinant human carbonic anhydrase I (hCA-I) by performing the fluorescence titration and steady-state kinetic experiments. The experimental data revealed that whereas the binding affinity of L1 for hCA-I was similar to that of benzenesulfonamide, the binding affinity of L2 was approximately 2 orders of magnitude higher, making L2 one of the most potent ligands or inhibitors of hCA-I. Since the enhanced binding or inhibitory potency of L2 is diminished (to the level of benzenesulfonamide) either in the presence of EDTA or upon treatment of the enzyme with diethyl pyrocarbonate, it is proposed that Cu(2+) of L2 interacts with one of the surface-exposed histidine residues of the enzyme. A cumulative account of the experimental data leads to the suggestion that the differential binding of L1 versus L2 to hCA-I is encoded in the chain length of the spacer moiety.

Acetazolamide↗
PubMed2005

Dental abnormalities in children preparing for pediatric bone marrow transplantation.

The purpose of our study was to describe the types and frequencies of altered dental development in pediatric patients preparing for bone marrow transplantation (BMT). Retrospective review of the medical records and panoramic radiographs of all patients who underwent BMT at St Jude Children's Research Hospital between 1990 and 2000 for whom pre-BMT dental examination and panoramic radiography records were available. All patients were treated on institutional protocols. We recorded patient demographics and radiographic evidence of microdontia, hypodontia, taurodontia, root stunting, caries, enamel pearls, and pulpal calcifications. The 259 patients identified (150 male and 109 female) had a median age of 12.82 years (range, 3.18-25.93 years) at the time of BMT. In total, 203 were Caucasian, 38 were African-American, and 18 were of other races. In all, 150 (57.9%) had abnormal dentition. The most common dental abnormalities were caries (n=84), pulpal calcifications (n = 34), and dental extractions (n = 33). Developmental abnormalities occurred less frequently: taurodontia (n = 8), hypodontia (n = 10), microdontia (n = 11), and root stunting (n = 11). Dental abnormalities are prevalent in children undergoing BMT. Pre-transplant oral hygiene and dental examination should be standard care in order to minimize potential sites of infection.

Adolescent↗
PubMed2005

Dental abnormalities after pediatric bone marrow transplantation.

Our purpose was to describe the types and frequencies of altered dental development in pediatric patients following bone marrow transplantation (BMT). A retrospective review of the medical records and panoramic radiographs of all patients who underwent BMT at St Jude Children's Research Hospital between 1990 and 2000, for whom pre-BMT and post-BMT dental examination and panoramic radiography records were available, is presented. All patients were treated on institutional protocols. We recorded patient demographics and radiographic evidence of microdontia, hypodontia, taurodontia, root stunting, caries, enamel pearls, dental restorations/extractions and pulpal calcification. The 99 patients identified (52 males, 47 females) had a median age of 13.5 years (range, 3.4-25.9 years) at the time of BMT. In all, 73 were Caucasian, 15 were African-American, and 11 were of other races. The frequency of radiographically evident root stunting in permanent teeth was significantly increased after BMT (P<0.001), but there was no significant change in the frequency of other dental abnormalities after BMT. Dental abnormalities are prevalent in survivors of childhood BMT, but only root stunting appeared to progress with BMT.

Adolescent↗
PubMed2005

Postprandial lipid abnormalities in type 2 diabetes mellitus.

AIM AND OBJECTIVES: To study the postprandial lipid abnormalities in patients with type 2 diabetes mellitus. MATERIAL AND METHODS: Postprandial lipids were studied in 20 male type 2 diabetic subjects (age 49.75 +/- 4.82 years) and 20 age and sex matched healthy controls (age 49.55 +/- 4.82 years) after an oral fat challenge which consisted of a meal providing 729 keal/m2 body surface area with 68 gm fat. RESULTS: Average duration of diabetes among diabetic was 2.32 +/- 3.03 years. The body mass index (cases 25.84 +/- 4.52; controls 25.74 +/- 5.0; p > 0.05) and waist-hip ratio (cases 1.06 +/- 0.13; controls 1.14 +/- 0.2; p > 0.05) were similar in both groups. While fasting serum lipids were not significantly different between the two groups, a number of serum lipid abnormalities were noted in type 2 diabetic subjects in the postprandial state. These included a higher triglyceride-area under curve (AUC) (cases 1298.08 +/- 485.2 vs. controls 922.15 +/- 390.47 mg/dl/8h; p=0.01), a higher triglyceride-area under incremental curve (AUIC) (cases 549.68 +/- 382.24; control 294.75 +/- 172.6 mg/dl/8h; p=0.01), a higher peak triglyceride level (cases 425.2 +/- 204.47 mg%, controls 283.9 +/- 11.6.94 mg%, p=0.01), a lower HDL-AUC (cases 130.35 +/- 33.55 vs. controls 168.48 +/- 56.01 mg/dl/8h, p=0.013) and a lower HDL nadir (Cases 28.05 +/- 10.94 mg%, controls 37.13 +/- 13.52 mg%, p < 0.02). Triglyceride AUC correlated significantly with fasting serum triglyceride (r=0.62) and BMI (r=0.7), but not with waist hip ratio or fasting serum insulin levels. Postprandial lipaemia did not correlate with age, duration of diabetes, fasting blood glucose or glycosylated hemoglobin. CONCLUSION: In conclusion, make type 2 diabetics demonstrate significant postprandial lipid abnormalities, particularly of triglycerides, which appear to be independent of glycaemic control.

Adult↗
PubMed2004

5-Hydroxydecanoate is metabolised in mitochondria and creates a rate-limiting bottleneck for beta-oxidation of fatty acids.

5-Hydroxydecanoate (5-HD) blocks pharmacological and ischaemic preconditioning, and has been postulated to be a specific inhibitor of mitochondrial ATP-sensitive K(+) (K(ATP)) channels. However, recent work has shown that 5-HD is activated to 5-hydroxydecanoyl-CoA (5-HD-CoA), which is a substrate for the first step of beta-oxidation. We have now analysed the complete beta-oxidation of 5-HD-CoA using specially synthesised (and purified) substrates and enzymes, as well as isolated rat liver and heart mitochondria, and compared it with the metabolism of the physiological substrate decanoyl-CoA. At the second step of beta-oxidation, catalysed by enoyl-CoA hydratase, enzyme kinetics were similar using either decenoyl-CoA or 5-hydroxydecenoyl-CoA as substrate. The last two steps were investigated using l-3-hydroxyacyl-CoA dehydrogenase (HAD) coupled to 3-ketoacyl-CoA thiolase. V(max) for the metabolite of 5-HD (3,5-dihydroxydecanoyl-CoA) was fivefold slower than for the corresponding metabolite of decanoate (l-3-hydroxydecanoyl-CoA). The slower kinetics were not due to accumulation of d-3-hydroxyoctanoyl-CoA since this enantiomer did not inhibit HAD. Molecular modelling of HAD complexed with 3,5-dihydroxydecanoyl-CoA suggested that the 5-hydroxyl group could decrease HAD turnover rate by interacting with critical side chains. Consistent with the kinetic data, 5-hydroxydecanoyl-CoA alone acted as a weak substrate in isolated mitochondria, whereas addition of 100 mum 5-HD-CoA inhibited the metabolism of decanoyl-CoA or lauryl-carnitine. In conclusion, 5-HD is activated, transported into mitochondria and metabolised via beta-oxidation, albeit with rate-limiting kinetics at the penultimate step. This creates a bottleneck for beta-oxidation of fatty acids. The complex metabolic effects of 5-HD invalidate the use of 5-HD as a blocker of mitochondrial K(ATP) channels in studies of preconditioning.

Animals↗
PubMed2004

Two-prong inhibitors for human carbonic anhydrase II.

The enzyme inhibitors are usually designed by taking into consideration the overall dimensions of the enzyme's active site pockets. This conventional approach often fails to produce desirable affinities of inhibitors for their cognate enzymes. To circumvent such constraints, we contemplated enhancing the binding affinities of inhibitors by attaching tether groups, which would interact with the surface exposed amino acid residues. This strategy has been tested for the inhibition of human carbonic anhydrase II. Benzenesulfonamide serves as a weak inhibitor for the enzyme, but when it is conjugated to iminodiacetate-Cu2+ (which interacts with the surface-exposed His residues) via a spacer group, its binding affinity is enhanced by about 2 orders of magnitude. This "two-prong" approach is expected to serve as a general strategy for converting weak inhibitors of enzymes into tight-binding inhibitors.

Binding Sites↗
PubMed2004

Protein surface-assisted enhancement in the binding affinity of an inhibitor for recombinant human carbonic anhydrase-II.

We elaborate on a novel strategy for enhancing the binding affinity of an active-site directed inhibitor by attaching a tether group, designed to interact with the surface-exposed histidine residue(s) of enzymes. In this approach, we have utilized the recombinant form of human carbonic anhydrase-II (hCA-II) as the enzyme source and benzenesulfonamide and its derivatives as inhibitors. The steady-state kinetic and the ligand binding data revealed that the attachment of iminodiacetate (IDA)-Cu(2+) to benzenesulfonamide (via a triethylene glycol spacer) enhanced its binding affinity for hCA-II by about 40-fold. No energetic contribution of either IDA or triethylene glycol spacer was found (at least in the ground state of the enzyme-inhibitor complex) when Cu(2+) was stripped off from the tether group-conjugated sulfonamide derivative. Arguments are presented that the overall strategy of enhancing the binding affinities of known inhibitors by attaching the IDA-Cu(2+) groups to interact with the surface-exposed histidine residues will find a general application in designing the isozyme-specific inhibitors as potential drugs.

Binding Sites↗
PubMed2004

Purification of recombinant human carbonic anhydrase-II by metal affinity chromatography without incorporating histidine tags.

Due to their involvement in diverse pathological conditions, carbonic anhydrases have been the targets of drug developments for the treatments of glaucoma, epilepsy, high altitude sickness, as well as cancer. Of about 14 isozymes of carbonic anhydrases, carbonic anhydrase-II (hCA-II) has been most extensively investigated from the structural, functional, and inhibitor design point of view. We discovered that hCA-II preferentially binds to the Sepharose-iminodiacetate (IDA)-Zn(2+) column, and such binding does not require incorporation of either N- or C-terminal histidine tags in the protein structure. By using the Sepharose-IDA-Zn(2+) affinity column, we purified the Escherichia coli expressed hCA-II with an overall recovery of 76%. The purified enzyme showed a single band on the SDS-PAGE. Due to ease in preparing the Sepharose-IDA-Zn(2+) column, and purifying hCA-II just in one step, the overall protocol will be ideal for producing bulk quantities of the enzyme for high throughput screening of inhibitors.

Binding Sites↗
PubMed2004

Bone mineral density and osteonecrosis in survivors of childhood allogeneic bone marrow transplantation.

Our purpose was to evaluate frequency and severity of bone mineral decrements and frequency of osteonecrosis in survivors of pediatric allogeneic bone marrow transplantation (alloBMT). We retrospectively reviewed demographic information, treatment, magnetic resonance (MR) imaging studies (hips and knees), and bone mineral density (BMD) studies of 48 patients as measured by quantitative computed tomography (QCT). In all, 24 patients were male; 37 were Caucasian. Median age at alloBMT was 10.3 years (1.6-20.4 years). Of the 48 patients, 43 underwent QCT. Median time between alloBMT and imaging was 5.1 years (1.0-10.2 years). Median BMD Z-score was -0.89 (-4.06 to 3.05). BMD Z-score tended to be associated with female sex (P=0.0559) but not with age at BMT, race, primary diagnosis, time from alloBMT, T-cell depletion of graft, total-body irradiation, or acute/chronic graft-versus-host disease (GVHD). MR showed osteonecrosis in 19 of 43 (44%). We found no associations between osteonecrosis and sex, race, diagnosis, age at BMT, history of GVHD, time from BMT, or T-cell depletion. Seven patients (15%) had MR changes of osteonecrosis and BMD Z-scores of less than -1 s.d. We conclude that pediatric alloBMT survivors have decreased BMD and are at risk of osteonecrosis. They should be monitored to assure early intervention that may ameliorate adverse outcomes.

Adolescent↗
PubMed2004

Encephalopathy in pediatric patients after allogeneic hematopoietic stem cell transplantation is associated with a poor prognosis.

SUMMARY: Encephalopathy is a poorly characterized complication of hematopoietic stem cell transplantation (HSCT). No comprehensive report of encephalopathy exists for children, and the literature contains only a few for adults. We analyzed a large cohort of 405 pediatric patients who underwent allogeneic HSCT during a 10-year period and identified 26 patients (6.4%) who experienced encephalopathy. Identifiable causes of encephalopathy included infection (n=5), single or multiorgan failure (n=4), medication-related complications (n=3), nonconvulsive seizures (n=4), acute disseminated encephalomyelitis (n=2), thrombotic thrombocytopenic purpura (n=2), and stroke (n=1). We were unable to identify the etiology of encephalopathy in five (19%) patients. The prognosis for pediatric patients with encephalopathy was poor: only four (15%) experienced complete neurologic recovery, and 10 (38%) patients experienced partial recovery with residual neurologic deficits. Nine (35%) patients with complete or partial recovery survive long term. A total of 17 patients died; one died of progressive encephalopathy, and 16 died of either relapse of primary disease or toxicity. MRI, CSF analysis including molecular testing for infectious pathogens, and brain biopsy were helpful in obtaining a diagnosis in most of our patients. However, a standardized approach to accurate and timely diagnosis and treatment is needed to improve outcome in these patients.

Adolescent↗