Raghu Pullakhandam

Binding and stabilization of transthyretin by curcumin

Biophysical evidences suggest that transthyretin (TTR) tetramer dissociation to the monomeric intermediate and subsequent polymerization leads to amyloid fibril formation, which is implicated in the pathogenesis of familial amyloid polyneuropathy (FAP) and senile systemic amyloidosis (SSA). Hence, inhibition of fibril formation is considered a potential therapeutic strategy. Here in we demonstrate that curcumin, a phenolic constituent of curry spice turmeric, binds to the active site of TTR through fluorescence quenching and ANS displacement studies. Binding of curcumin appears to inhibit the denaturant induced tertiary and quaternary structural changes in TTR as monitored by intrinsic emission fluorescence and glutaraldehyde cross-linking studies. However, curcumin did not bind to TTR at acidic pH. Protonation/ isomerization of the side chain oxygen atoms of curcumin at low pH might hamper the binding. These results suggest that curcumin binds to and stabilizes TTR thereby highlight the importance of the side chain conformations of the ligand in binding to TTR.

Exome Analysis Identified a Novel Mutation in the RBP4 Gene in a Consanguineous Pedigree with Retinal Dystrophy and Developmental Abnormalities

Retinitis Pigmentosa (RP) is a common form of retinal degeneration characterized by photoreceptor degeneration and retinal pigment epithelium (RPE) atrophy causing loss of visual field and acuities. Exome sequencing identified a novel homozygous splice site variant (c.111+1G>A) in the gene encoding retinol binding protein 4 (RBP4). This change segregated with early onset, progressive, and severe autosomal recessive retinitis pigmentosa (arRP) in an eight member consanguineous pedigree of European ancestry. Additionally, one patient exhibited developmental abnormalities including patent ductus arteriosus and chorioretinal and iris colobomas. The second patient developed acne from young age and extending into the 5th decade. Both patients had undetectable levels of RBP4 in the serum suggesting that this mutation led to either mRNA or protein instability resulting in a null phenotype. In addition, the patients exhibited severe vitamin A deficiency, and diminished serum retinol levels. Circulating transthyretin levels were normal. This study identifies the RBP4 splice site change as the cause of RP in this pedigree. The presence of developmental abnormalities and severe acne in patients with retinal degeneration may indicate the involvement of genes that regulate vitamin A absorption, transport and metabolism.

Zinc inhibits oxidative stress-induced iron signaling and apoptosis in Caco-2 cells

Studies in humans and animals have suggested negative interactions of iron and zinc during their intestinal absorption. Further, zinc seems to prevent iron-induced oxidative damage in rats, which was hypothesized to be through the modulation of the intracellular iron signaling pathway. The aim of this study was, therefore, to understand the effects of zinc on oxidant-induced iron signaling and cell death in human enterocyte-like Caco-2 cells. We demonstrate that zinc decreases glucose/glucose oxidase (H(2)O(2)-generating system)-induced iron uptake and inhibits iron-regulatory protein 1 activation and divalent metal ion transporter 1 expression. There was also a concomitant decrease in oxidant-induced intracellular labile iron and restoration of ferritin and metallothionein expression. Further, zinc enhanced the Bcl-2/Bax ratio and reduced caspase-3 activity, leading to inhibition of apoptosis. Interestingly, bathophenanthroline disulfonic acid, an extracellular iron chelator, emulated the effects of zinc except for the reduced ferritin levels. These results suggest that zinc inhibits apoptosis by reducing oxidant-induced iron signaling in Caco-2 cells.

Thiamine reduces tissue lead levels in rats: mechanism of interaction

Lead (Pb) toxicity has been a serious concern in industrialized societies because of its association with functional deficits in nervous, haematopoietic and renal systems. Several studies have shown beneficial effects of thiamine on Pb toxicity. It is speculated that Pb chelation by thiamine may be a possible mechanism. However, the exact nature of these interactions remained elusive. In the present study we have characterized the interaction of Pb with thiamine using UV–Vis as well as fluorescence spectroscopic methods and studied the effect of thiamine treatment on blood and tissue Pb levels during simultaneous or post-exposure to Pb in rat model. The spectroscopic studies revealed that Pb interacts with the pyrimidine ring of thiamine, leading to its solubilization at physiological pH. Further, thiamine reduced the Pb levels in blood, kidney and bone during both simultaneous and post-exposure Pb treatment. Interestingly, thiamine appears to prevent the accumulation of Pb in bone during simultaneous treatment. Together these results suggest that pyrimidine ring of thiamine mediates its interaction with Pb, leading to the prevention of its accumulation and/or increased clearance from tissues.

Increased risk of cataract development in WNIN‐obese rats due to accumulation of intralenticular sorbitol

Epidemiological studies have reported an association between obesity and increased incidence of ocular complications including cataract, yet the underlying biochemical and molecular mechanisms remained unclear. Previously we had demonstrated accumulation of sorbitol in the lens of obese rats (WNIN/Ob) and more so in a related strain with impaired glucose tolerance (WNIN/GR‐Ob). However, only a few (15–20%) WNIN/Ob and WNIN/GR‐Ob rats develop cataracts spontaneously with age. To gain further insights, we investigated the susceptibility of eye lens proteins of these obese rat strains to heat‐ and UV‐induced aggregation in vitro, lens opacification upon glucose‐mediated sorbitol accumulation ex vivo, and onset and progression of cataract was followed by galactose feeding and streptozotocin (STZ) injection. The results indicated increased susceptibility toward heat‐ or UV‐induced aggregation of lens proteins in obese animals compared to their littermate lean controls. Further, in organ culture studies glucose‐induced sorbitol accumulation was found to be higher and thus the lens opacification was faster in obese animals compared to their lean littermates. Also, the onset and progression of galactose‐ or STZ‐induced cataractogenesis was faster in obese animals compared to lean control. These results together with our previous observations suggest that obesity status could lead to hyperaccumulation of sorbitol in eye lens, predisposing them to cataract, primarily by increasing their susceptibility to environmental and/or physiological factors. Further, intralenticular sorbitol accumulation beyond a threshold level could lead to cataract in WNIN/Ob and WNIN/GR‐Ob rats.

Lead and essential trace element levels in school children: A cross-sectional study

Background: Nutritional status is thought to modulate susceptibility to lead (Pb) toxicity. The type and nature of these interactions needs to be investigated. Aim: To assess the prevalence of sub-clinical Pb toxicity (defined by ≥ 10 μ_rm;g/dL blood levels) and trace element deficiencies (Iron (Fe), Zinc (Zn), Copper (Cu) and Magnesium (Mg)) and to find out their possible relationship, if any. Subjects and methods: A cross-sectional survey was carried out in a total of 195 school children residing in urban (n = 65), urban heavy traffic (n = 65) and urban industrial (n = 65) zones of Hyderabad, India. Blood Pb, trace element levels, haemoglobin and δ-aminolevulinate dehydratase (δ-ALAD) activity was measured. Results: High blood lead levels ( ≥ 10 μ_rm;g/dL) were observed in 54.3% of children while percentage prevalence of trace element deficiencies such as Fe (16.2%), Zn (68.6%), Mg (41.7%) and Cu (25%) were also high in children included in the study. Higher blood Pb levels and reduced δ-ALAD activity was observed in children residing in heavy traffic and industrial areas. Blood Pb levels but not δ-ALAD activity correlated inversely with serum Fe in heavy traffic and industrial children, respectively. Interestingly, δ-ALAD activity but not blood Pb levels correlated inversely with trace element levels only in urban children. Conclusions: These results suggest higher prevalence of sub-clinical Pb toxicity and trace element deficiencies in urban children. Further, high blood Pb levels appear to be correlated with reduced δ-ALAD activity and iron status in Pb exposed children.

Ferric reductase activity of low molecular weight human milk fraction is associated with enhanced iron solubility and uptake in Caco-2 cells

It is known that the fractional absorption of extrinsic iron from human milk is higher in infants and adults. A low molecular weight milk fraction has been proposed to increase the bioavailability of iron from human milk. Nevertheless, the mechanisms remained elusive. Here in we demonstrate ferric reductase activity (Km7.73x10(-6)M) in low molecular weight human milk fraction (10kF, filtrate derived from ultra filtration of milk whey through 10kDa cutoff membrane), which increased ferric iron solubility and iron uptake in Caco-2 cells. The 10kF fraction was as effective as ascorbic acid (1:20 iron to ascorbic acid) in increasing the ferric iron solubility and uptake in Caco-2 cells. Further, gel filtration chromatography on peptide column led to co-elution of ferric reductase and iron solubilization activities at an apparent molecular mass of <1500Da. Interestingly, only these fractions containing ferric reductase activity also stimulated the uptake of iron in Caco-2 cells. Thus, it is concluded that human milk possesses ferric reductase activity and is associated with ferric iron solubilization and enhanced absorption.

Herbs as a dietary source of iron

Purpose – The purpose of this paper is to screen for iron bioavailability and absorption-promoting activity in selected herbs. Evidence is needed to promote and practice food-based strategies such as use of plants or their parts for treating iron deficiency anemia. Design/methodology/approach – Eight Indian herbs, considered to be iron rich and/or hematinic, namely, Boerhavia diffusa, Trachyspermum ammi, Amaranthus paniculatus, Lepidium sativum, Medicago sativa, Asparagus racemosus, Sesamum indicum and Piper longum, were selected. Their mineral composition and phytate and tannin contents were analyzed. Endogenous iron bioavailability was assessed in human enterocyte cell line model, Caco-2 cells, using cellular ferritin induction. Iron absorption-promoting activity was tested similarly in two herbs and their mineral extract by the addition of exogenous iron or ascorbic acid. Findings – Based on compositional analysis, B. diffusa, L. sativum and T. ammi had high iron (> 40 mg/100 g) and tannin/phytate. A. ...