Carboxymethyl lysine induces EMT in podocytes through transcription factor ZEB2: Implications for podocyte depletion and proteinuria in diabetes mellitus.

Advanced glycation end-products (AGEs) are implicated in the pathogenesis of diabetic nephropathy (DN). N-carboxymethyl-lysine (CML) is one of the predominant AGEs that accumulate in all renal compartments of diabetic patients. Nevertheless, the direct effect of CML on podocyte biology has not been explored. In this study, we demonstrate the induction of the transcription factor Zeb2 in podocytes upon exposure to CML through activation of NF-kB signaling cascade. Zeb2 orchestrates epithelial-mesenchymal transformation (EMT), during which cell-cell and cell-extracellular matrix interactions are feeble and enable epithelial cells to become invasive. CML treatment induced both NF-kB and Zeb2 promoter activity and suppressed E-cadherin promoter activity. Inhibition of NF-kB activity prevented CML dependent induction of Zeb2 and loss of E-cadherin. While the exposure of podocytes to CML results in increased podocyte permeability, shRNA-mediated knockdown of Zeb2 expression abrogated CML-mediated podocyte permeability. Further, in vivo findings of elevated CML levels concurrent with increased expression of ZEB2 in glomeruli and proteinuria in diabetic rats confirm that CML-mediated manifestations in the kidney under chronic diabetes conditions. These in vitro and in vivo results envisage the novel axis of NFkB-ZEB2 in podocytes playing a significant role in eliciting EMT and pathogenesis of DN.

Studying nanoparticles' 3D shape by aspect maps: Determination of the morphology of bacterial magnetic nanoparticles.

Magnetic nanoparticles (MNPs) are widely investigated due to their potential use in various applications, ranging from electronics to biomedical devices. The magnetic properties of MNPs are strongly dependent on their size and shape (i.e., morphology), thus appropriate tools to investigate their morphology are fundamental to understand the physics of these systems. Recently a new approach to study nanoparticle morphology by Transmission Electron Microscopy (TEM) analysis has been proposed, introducing the so-called Aspect Maps (AMs). In this paper, a further evolution of the AM method is presented, allowing determination of the nanoparticles' 3D shape by TEM image. As a case study, this paper will focus on magnetite nanoparticles (Fe3O4), with a mean size of ∼45 nm extracted from Magnetospirillum gryphiswaldense magnetostatic bacteria (MTB). The proposed approach gives a complete description of the nanoparticles' morphology, allowing estimation of an average geometrical size and shape. In addition, preliminary investigation of the magnetic properties of MTB nanoparticles was performed, giving some insight into interparticle interactions and on the reversal mechanism of the magnetization.

Growth Hormone Induces Transforming Growth Factor-Beta-Induced Protein in Podocytes: Implications for Podocyte Depletion and Proteinuria.

The glomerular podocytes form a major size selective barrier for the filtration of serum proteins and reduced podocyte number is a critical event in the pathogenesis of proteinuria during diabetic nephropathy (DN). An elevated level of growth hormone (GH) is implicated as a causative factor in the development of nephropathy in patients with type 1 diabetes mellitus. We have previously shown that podocytes express GH receptor and are a target for GH action. To elucidate the molecular basis for the effects of GH on podocyte depletion, we conducted PCR-array analyses for extracellular matrix and adhesion molecules in podocytes. Our studies reveal that GH increases expression of a gene that encodes transforming growth factor-beta-induced protein (TGFBIp) expression. Similarly, microarray data retrieved from the Nephromine database revealed elevation of TGFBIp in patients with DN. Treatment with GH results in increased secretion of extracellular TGFBIp by podocytes. Both GH and TGFBIp induced apoptosis and epithelial mesenchymal transition (EMT) of podocytes. Exposure of podocytes to GH and TGFBIp resulted in increased migration of cells and altered podocyte permeability to albumin across podocyte monolayer. Administration of GH to rats induced EMT and apoptosis in the glomerular fraction of the kidney. Therefore, we conclude that the GH-dependent increase in TGFBIp in the podocyte is one of the mechanisms responsible for podocyte depletion in DN.

Targeted deletion of growth hormone (GH) receptor in macrophage reveals novel osteopontin-mediated effects of GH on glucose homeostasis and insulin sensitivity in diet-induced obesity.

We investigated GH action on macrophage (MΦ) by creating a MΦ-specific GH receptor-null mouse model (MacGHR KO). On a normal diet (10% fat), MacGHR KO and littermate controls exhibited similar growth profiles and glucose excursions on intraperitoneal glucose (ipGTT) and insulin tolerance (ITT) tests. However, when challenged with high fat diet (HFD, 45% fat) for 18 weeks, MacGHR KO mice exhibited impaired ipGTT and ITT compared with controls. In MacGHR KO, adipose-tissue (AT) MΦ abundance was increased with skewing toward M1 polarization. Expression of pro-inflammatory cytokines (IL1ß, TNF-α, IL6, and osteopontin (OPN)) were increased in MacGHR KO AT stromal vascular fraction (SVF). In MacGHR KO AT, crown-like-structures were increased with decreased insulin-dependent Akt phosphorylation. The abundance of phosphorylated NF-κB and of OPN was increased in SVF and bone-marrow-derived MΦ in MacGHR KO. GH, acting via an NF-κB site in the distal OPN promoter, inhibited the OPN promoter. Thus in diet-induced obesity (DIO), lack of GH action on the MΦ exerts an unexpected deleterious effect on glucose homeostasis by accentuating AT inflammation and NF-κB-dependent activation of OPN expression. These novel results in mice support the possibility that administration of GH could have salutary effects on DIO-associated chronic inflammation and insulin resistance in humans.

Hungatella effluvii gen. nov., sp. nov., an obligately anaerobic bacterium isolated from an effluent treatment plant, and reclassification of Clostridium hathewayi as Hungatella hathewayi gen. nov., comb. nov.

A Gram-stain-positive, rod-shaped, spore-forming and strictly anaerobic bacterium, designated UB-B.2(T), was isolated from an industrial effluent anaerobic digester sample. It grew optimally at 30 °C and pH 7.0. Comparative analysis of the 16S rRNA gene sequence confirmed that strain UB-B.2(T) was closely related to Clostridium hathewayi DSM 13479(T) (97.84% similarity), a member of rRNA gene cluster XIVa of the genus Clostridium, and formed a coherent cluster with other related members of the Blautia (Clostridium) coccoides rRNA group in phylogenetic analyses. The end products of glucose fermentation by strain UB-B.2(T) were acetate and propionate. The G+C content of the DNA was 51.4 mol%. Although strain UB-B.2(T) showed 97.8% 16S rRNA gene sequence identity to the type strain of C. hathewayi, it exhibited only 38.4% relatedness at the whole-genome level. It also showed differences from its closest phylogenetic relative, C. hathewayi DSM 13479(T), in phenotypic characteristics such as hydrolysis of aesculin, starch and urea and fermentation end products. Both strains showed phenotypic differences from the members of rRNA gene cluster XIVa of the genus Clostridium. Based on these differences, C. hathewayi DSM 13479(T) and strain UB-B.2(T) were identified as representatives of a new genus of the family Clostridiaceae. Thus, we propose the reclassification of Clostridium hathewayi as Hungatella hathewayi gen. nov., comb. nov., the type species of the new genus (type strain DSM 13479(T) = CCUG 43506(T) = MTCC 10951(T)). Strain UB-B.2(T) ( = MTCC 11101(T) = DSM 24995(T)) is assigned to the novel species Hungatella effluvii gen. nov., sp. nov as the type strain.

Metabolic syndrome and associated chronic kidney diseases: nutritional interventions.

Lifestyle changes such as dietary habits, sedentary life and consumption of energy-dense foods that have occurred over the years has led to an epidemic of abdominal obesity, which in turn resulted in dramatic increase in the prevalence of metabolic syndrome (MetS). Different expert panels have provided various definitions for MetS to enable a clinical diagnosis and treatment of patients at risk of associated complications. Obesity and obesity mediated MetS has been paralleled by escalation in the incidence of chronic kidney disease (CKD). A better understanding of the pathophysiology of MetS and identification of individuals with MetS early in the life course could be important for initiating interventions such as lifestyle modification and dietary restrictions that form the basis for prevention and treatment of MetS and related co-morbidities including CKD. This review is intended to provide a comprehensive summary of the evolution of definition of MetS and association of MetS with CKD. In particular, mechanism of obesity and diabetes mediated CKD and emerging dietary therapies for MetS associated CKD will be discussed.

Flavobacterium nitratireducens sp. nov., an amylolytic bacterium of the family Flavobacteriaceae isolated from coastal surface seawater.

A novel Gram-negative, rod-shaped, non-motile bacterium, designated strain N1(T), was isolated from a marine water sample collected from the sea shore, Bay of Bengal, Visakhapatnam, India. The strain was positive for starch hydrolysis, nitrate reduction and ornithine decarboxylase activities and negative for citrate utilization, urease, oxidase, catalase and DNase activities. The predominant fatty acids were C16 : 0 3-OH, iso-C15 : 0, iso-C15 : 0 3-OH, iso-C17 : 0 3-OH, anteiso-C15 : 0, C16 : 0, C15 : 0 3-OH, and C16 : 1ω7c and/or iso-C15 : 0 2-OH (summed feature 3). Strain N1(T) contained menaquinone 6 (MK-6) as the sole respiratory quinone. The only polyamine was homospermidine and the major polar lipids were phosphatidylethanolamine (PE), three unidentified aminolipids (AL1-AL3) and two unidentified lipids (L1, L2). The DNA G+C content of the strain was 36.3 mol%. 16S rRNA gene sequence analysis indicated that strain N1(T) was a member of the genus Flavobacterium and closely related to Flavobacterium resistens with pairwise sequence similarity of 96.5 %. Phylogenetic analysis showed that strain N1(T) clustered with Flavobacterium glycines and Flavobacterium daejeonense with a distance of 4.8 and 6.0 % (95.2 and 94.0 % similarity), respectively. Based on the phenotypic characteristics and on phylogenetic inference, strain N1(T) represents a novel species of the genus Flavobacterium, for which the name Flavobacterium nitratireducens sp. nov. is proposed. The type strain is N1(T) ( = MTCC 11155(T) = JCM 17678(T)).

Caldimonas meghalayensis sp. nov., a novel thermophilic betaproteobacterium isolated from a hot spring of Meghalaya in northeast India.

While studying the microbial diversity of hot springs of North-east India we isolated a strain AK31T from the Jakrem hot spring of Meghalaya. The strain formed light yellow colonies on nutrient agar and was Gram negative, non spore-forming rods, motile with single polar flagellum. The strain was positive for oxidase and catalase and hydrolysed starch and weakly urea. The predominant cellular fatty acids were C16:0 (34.8 %), C17:0 cyclo (27.1 %), C16:1 ω7c and/or iso-C15:0 2OH (summed feature 3) (9.6 %), C10:0 3OH (8.0 %), C12:0 (5.8 %), C14:0 (5.3 %) and C18:1 ω7c (5.3 %). Strain AK31T contained ubiquinone-8 as the major respiratory quinone and diphosphatidylglycerol, phosphatidylethanolamine, three unidentified phospholipids and one unidentified glycolipid as the polar lipids. The G + C content of the DNA of the strain AK31T was 66.7 mol%. The 16S rRNA gene sequence analysis indicated that strain AK31T was member of the genus Caldimonas and closely related to Caldimonas manganoxidans JCM 10698T and Caldimonas taiwanensis On1T with 96.9 % similarity and with Aquincola tertiaricarbonis L10T and Azohydromonas australica IAM 12664T with 96.5 and 96.4 % similarity respectively. Phylogenetic analyses indicated that the strain AK31T clustered with C. manganoxidans JCM 10698T and C. taiwanensis On1T with a phylogenetic distance of 3.25 %. Based on data from the current polyphasic study, strain AK31T is proposed as a novel species of the genus Caldimonas, for which the name Caldimonas meghalayensis sp. nov. is proposed. The type strain of C. meghalayensis is AK31T (= MTCC 11703T = JCM 18786T).

Lutibaculum baratangense gen. nov., sp. nov., a proteobacterium isolated from a mud volcano.

A novel Gram-negative, oval to rod-shaped, motile bacterium, strain AMV1(T), was isolated from a soil sample collected from a mud volcano of Baratang Island, Andamans, India. The predominant fatty acids were C(16:0) (5.7%), C(18:1)ω7c (78.6%) and C(19:0) cyclo ω8c (6.3%). Strain AMV1(T) contained ubiquinone 10 (Q-10) as the major respiratory quinone and minor quantities of ubiquinone 9 (Q-9). The polar lipids consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, three unidentified lipids, one unidentified phospholipid and one unidentified aminolipid. 16S rRNA gene sequence analysis indicated that strain AMV1(T) was related most closely to the type strains of Tepidamorphus gemmatus, Bauldia consociata, Afifella pfennigii and Amorphus coralli, four members of the order Rhizobiales (class Alphaproteobacteria), with pairwise sequence similarities of 95.0, 94.5, 94.4 and 94.0%, respectively; it shared <94% 16S rRNA gene sequence similarity with all the other members of the order Rhizobiales. Phylogenetic analyses indicated that strain AMV1(T) clustered with Tepidamorphus gemmatus and with species of the genera Amorphus, Rhodobium and Afifella. Phenotypic and phylogenetic characteristics thus suggest that strain AMV1(T) is a representative of a novel species of a new genus, for which the name Lutibaculum baratangense gen. nov., sp. nov. is proposed. The type strain of Lutibaculum baratangense is AMV1(T) ( = KCTC 22669(T) = NBRC 105799(T) = CCUG 58046(T)).

Growth hormone (GH)-dependent expression of a natural antisense transcript induces zinc finger E-box-binding homeobox 2 (ZEB2) in the glomerular podocyte: a novel action of gh with implications for the pathogenesis of diabetic nephropathy.

Growth hormone (GH) excess results in structural and functional changes in the kidney and is implicated as a causative factor in the development of diabetic nephropathy (DN). Glomerular podocytes are the major barrier to the filtration of serum proteins, and altered podocyte function and/or reduced podocyte number is a key event in the pathogenesis of DN. We have previously shown that podocytes are a target for GH action. To elucidate the molecular basis for the effects of GH on the podocyte, we conducted microarray and RT-quantitative PCR analyses of immortalized human podocytes and identified zinc finger E-box-binding homeobox 2 (ZEB2) to be up-regulated in a GH dose- and time-dependent manner. We established that the GH-dependent increase in ZEB2 levels is associated with increased transcription of a ZEB2 natural antisense transcript required for efficient translation of the ZEB2 transcript. GH down-regulated expression of E- and P-cadherins, targets of ZEB2, and inhibited E-cadherin promoter activity. Mutation of ZEB2 binding sites on the E-cadherin promoter abolished this effect of GH on the E-cadherin promoter. Whereas GH increased podocyte permeability to albumin in a paracellular albumin influx assay, shRNA-mediated knockdown of ZEB2 expression abrogated this effect. We conclude that GH increases expression of ZEB2 in part by increasing expression of a ZEB2 natural antisense transcript. GH-dependent increase in ZEB2 expression results in loss of P- and E-cadherins in podocytes and increased podocyte permeability to albumin. Decreased expression of P- and E-cadherins is implicated in podocyte dysfunction and epithelial-mesenchymal transition observed in DN. We speculate that the actions of GH on ZEB2 and P- and E-cadherin expression play a role in the pathogenesis of microalbuminuria of DN.

Identification of fruity aroma-producing compounds from Chryseobacterium sp. isolated from the Western Ghats, India.

A fruity aroma-producing strain WG4 was isolated from a water sample collected from the Western Ghats, India. The 16S rRNA gene sequence analysis of strain WG4 indicated that Chryseobacterium indologenes, a member of the family 'Flavobacteriaceae' is the closest related species with a pair-wise sequence similarity of 98.6%. Strain WG4 produces a fruity aroma when grown on nutrient or trypticase soy agar plates. The fruity aroma is more when the strain WG4 is grown on agar plates compared to their growth in broth. The aromatic compounds produced by the strain WG4 were identified as ester compounds and were confirmed as ethyl-2-methylbutyrate and ethyl-3-methylbutyrate based on Gas Chromatography-Mass Spectrometry (GC-MS) analysis and using standard reference compounds. Even after repeated subcultures strain WG4 produced the same aroma in high intensity. Thus, strain WG4 could serve as a source for the production of these flavour compounds.

Retraction. Cecembia lonarensis gen. nov., sp. nov., a novel haloalkalitolerant bacterium of the family Cyclobacteriaceae, isolated from a haloalkaline lake and emended descriptions of the genera Indibacter, Nitritalea, Belliella and Aquiflexum.

The authors would like to retract this publication in order to improve the chemotaxonomic data as it has become apparent that there are inconsistencies in the data presented in this paper. This paper has been retracted from IJSEM Papers in Press and will not be published in print in IJSEM.

Leptin induced in vitro development of ovarian follicles in sheep is related to the expression of P450 aromatase and steroidogenesis.

The objective of this study was to test the hypothesis that Leptin induced in vitro growth in preantral follicles in sheep involves modulation of P450 aromatase expression and steroidogenesis. Accordingly, the expression of P450 aromatase gene was studied in the cumulus cells and oocytes isolated from different stages of preantral follicles (PFs') grown in vivo, cultured in TCM 199B, TCM 199B + Leptin (10 ng/ml) (TCM199BL) or a standard PF culture medium supplemented with Leptin (10 ng/ml) (SML). Ovarian follicles grown in vivo or in SML expressed P450 aromatase both in cumulus cells and oocytes at all the development stages. In the oocytes from PFs' grown in vitro, P450 expression was consistently lower than in those from in vivo grown follicles at all except the preantral stage. The patterns of expression of aromatase gene in the cumulus cells from in vivo grown and the PFs' cultured in TCM 199BL were similar. Significantly higher levels of progesterone production were supported by SML at all the development stages than the other two media. Oestradiol concentration in the spent TCM 199B and SML showed a significant increase as the development progressed from preantral to large antral stage. However, such increase was not sustained beyond early antral stage in the PFs' cultured in TCM199BL. It is concluded that Leptin modulates the expression P450 aromatase while supporting the in vitro development of the ovarian follicles in sheep.

Revisiting Goodenough-Kanamori rules in a new series of double perovskites LaSr1-xCaxNiReO6.

The magnetic ground states in highly ordered double perovskites LaSr1-xCaxNiReO6 (x = 0.0, 0.5, 1.0) are studied in view of the Goodenough-Kanamori rules of superexchange interactions in this paper. In LaSrNiReO6, Ni and Re sublattices are found to exhibit curious magnetic states separately, but no long range magnetic ordering is achieved. The magnetic transition at ~255 K is identified with the independent Re sublattice magnetic ordering. Interestingly, the sublattice interactions are tuned by modifying the Ni-O-Re bond angles through Ca doping. Upon Ca doping, the Ni and Re sublattices start to display a ferrimagnetically ordered state at low temperature. The neutron powder diffraction data reveals long range ferrimagnetic ordering of the Ni and Re magnetic sublattices along the crystallographic b-axis. The transition temperature of the ferrimagnetic phase increases monotonically with increasing Ca concentration.

Effect of glycation on alpha-crystallin structure and chaperone-like function.

The chaperone-like activity of alpha-crystallin is considered to play an important role in the maintenance of the transparency of the eye lens. However, in the case of aging and in diabetes, the chaperone function of alpha-crystallin is compromized, resulting in cataract formation. Several post-translational modifications, including non-enzymatic glycation, have been shown to affect the chaperone function of alpha-crystallin in aging and in diabetes. A variety of agents have been identified as the predominant sources for the formation of AGEs (advanced glycation end-products) in various tissues, including the lens. Nevertheless, glycation of alpha-crystallin with various sugars has resulted in divergent results. In the present in vitro study, we have investigated the effect of glucose, fructose, G6P (glucose 6-phosphate) and MGO (methylglyoxal), which represent the major classes of glycating agents, on the structure and chaperone function of alpha-crystallin. Modification of alpha-crystallin with all four agents resulted in the formation of glycated protein, increased AGE fluorescence, protein cross-linking and HMM (high-molecular-mass) aggregation. Interestingly, these glycation-related profiles were found to vary with different glycating agents. For instance, CML [N(epsilon)-(carboxymethyl)lysine] was the predominant AGE formed upon glycation of alpha-crystallin with these agents. Although fructose and MGO caused significant conformational changes, there were no significant structural perturbations with glucose and G6P. With the exception of MGO modification, glycation with other sugars resulted in decreased chaperone activity in aggregation assays. However, modification with all four sugars led to the loss of chaperone activity as assessed using an enzyme inactivation assay. Glycation-induced loss of alpha-crystallin chaperone activity was associated with decreased hydrophobicity. Furthermore, alpha-crystallin isolated from glycated TSP (total lens soluble protein) had also increased AGE fluorescence, CML formation and diminished chaperone activity. These results indicate the susceptibility of alpha-crystallin to non-enzymatic glycation by various sugars and their derivatives, whose levels are elevated in diabetes. We also describe the effects of glycation on the structure and chaperone-like activity of alpha-crystallin.

Effects of at-home and in-office bleaching agents on the shear bond strength of metal, ceramic, and composite brackets to enamel.

AIMS AND OBJECTIVES: This study aimed to evaluate the effects of at-home and in-office bleaching on the shear bond strength (SBS) of metal, ceramic, and composite orthodontic brackets and to compare their SBSs. SUBJECTS AND METHODS: A total of 96 human lower premolar teeth were used for this study. Six teeth were used for scanning electron microscopic study while the remaining ninety were divided into three equal groups. Each group was further subdivided into three subgroups with ten samples each. Three protocols were used. In the at-home bleaching group (n = 30), opalescence non-PF (potassium nitrate and fluoride) bleaching agent (10% carbamide peroxide) was applied onto the teeth daily for 14 days and left for 8 h each day. Teeth in the in-office group (n = 30) were treated twice in consecutive days with Opalescence boost PF (40% hydrogen peroxide). After bleaching, the specimens were stored in distilled water for 1 day before bonding. SBS testing was performed on all teeth using Instron universal testing machine. RESULTS: Analysis of variance indicated a significant difference (P < 0.005) among the groups. Maximum SBS was shown by ceramic brackets in control group (Ib) and minimum was shown by composite brackets of in-office bleached group (IIIc). CONCLUSIONS: The results showed that at-home bleaching did not affect the SBS significantly whereas in-office bleaching reduced SBS of metal, ceramic, and composite brackets significantly. It is preferable to use metal or ceramic brackets than composite brackets for bonding 24 h after bleaching.

Recipe for High Moment Materials with Rare-earth and 3d Transition Metal Composites.

Materials with high volume magnetization are perpetually needed for the generation of sufficiently large magnetic fields by writer pole of magnetic hard disks, especially for achieving increased areal density in storage media. In search of suitable materials combinations for this purpose, we have employed density functional theory to predict the magnetic coupling between iron and gadolinium layers separated by one to several monolayers of 3d transition metals (Sc-Zn). We demonstrate that it is possible to find ferromagnetic coupling for many of them and in particular for the early transition metals giving rise to high moment. Cr and Mn are the only elements able to produce a significant ferromagnetic coupling for thicker spacer layers. We also present experimental results on two trilayer systems Fe/Sc/Gd and Fe/Mn/Gd. From the experiments, we confirm a ferromagnetic coupling between Fe and Gd across a 3 monolayers Sc spacer or a Mn spacer thicker than 1 monolayer. In addition, we observe a peculiar dependence of Fe/Gd magnetic coupling on the Mn spacer thickness.

Ellagic acid inhibits non-enzymatic glycation and prevents proteinuria in diabetic rats.

The formation of advanced glycation end products (AGEs) is a characteristic feature of diabetic tissues and accumulation of these products has been implicated in the pathogenesis of micro- and macrovascular complications of diabetes including diabetic nephropathy (DN). Compelling evidence suggests that AGEs mediate progressive alteration in the renal architecture and loss of renal function whereas inhibitors of AGEs prevent the progression of experimental DN. We have investigated the potential of ellagic acid (EA), a polyphenol present abundantly in fruits and vegetables, to prevent in vivo accumulation of AGE and to ameliorate renal changes in diabetic rats. Streptozotocin-induced diabetic rats were fed with either 0.2% or 2% of EA in the diet for 12 weeks. Dietary supplementation of EA to diabetic rats prevented the glycation mediated RBC-IgG-cross-links and HbA1c accumulation. EA inhibited the accumulation of N-carboxymethyl lysine (CML), a predominant AGE in the diabetic kidney. Further, EA also prevented the AGE-mediated loss of expression of podocyte slit diaphragm proteins: nephrin and podocin. By inhibiting CML formation, EA improved renal function in rats as evidenced by urinary albumin and creatinine levels. In conclusion, EA inhibited AGE accumulation in the diabetic rat kidney and ameliorated AGE-mediated pathogenesis of DN.

Modulation of alpha-crystallin chaperone activity in diabetic rat lens by curcumin.

PURPOSE: A decline in the chaperone-like activity of eye lens alpha-crystallin in diabetic conditions has been reported. In this study, we investigated whether curcumin, a dietary antioxidant, can manipulate the chaperone-like activity of alpha-crystallin in diabetic rat lens. METHODS: A group of rats received ip injection of streptozotocin (STZ; 35 mg/kg body weight in buffer) to induce hyperglycemia, while another group of rats received only buffer as vehicle and served as control. STZ-treated rats were assigned to 3 groups and fed either no curcumin or 0.002% or 0.01% curcumin, respectively. Cataract progression due to hyperglycemia was monitored with a slit lamp biomicroscope. At the end of 8 weeks animals were sacrificed and lenses were collected. alphaH- and alphaL-crystallins from a set of pooled lenses in each group were isolated by gel filtration. Chaperone activity, hydrophobicity, and secondary and tertiary structure of alphaH- and alphaL-crystallins were assessed by light scattering/spectroscopic methods. RESULTS: A decrease in chaperone-like activity of alphaH- and alphaL-crystallins was observed in STZ-treated diabetic rats. The declined chaperone-like activity due to hyperglycemia was associated with reduced hydrophobicity and altered secondary and tertiary structure of alphaH- and alphaL-crystallins. Interestingly, alphaH- and alphaL-crystallins isolated from curcumin fed diabetic rat lenses had shown improved chaperone-like activity as compared to alphaH- and alphaL-crystallins from untreated diabetic rat lens. Feeding of curcumin prevented the alterations in hydrophobicity and structural changes due to STZ-induced hyperglycemia. Modulation of functional and structural properties by curcumin was found to be greater with the alphaL-crystallin than alphaH-crystallin. Loss of chaperone activity of alpha-crystallin, particularly alphaL-crystallin, in diabetic rat lens could be attributed at least partly to increased oxidative stress. Being an antioxidant, curcumin feeding has prevented the loss of alpha-crystallin chaperone activity and delayed the progression and maturation of diabetic cataract. CONCLUSIONS: We demonstrate that curcumin, at the levels close to dietary consumption, prevented the loss of chaperone-like activity of alpha-crystallin vis-a-vis cataractogenesis due to diabetes in rat lens.

Effect of dicarbonyl-induced browning on alpha-crystallin chaperone-like activity: physiological significance and caveats of in vitro aggregation assays.

Alpha-crystallin is a member of the small heat-shock protein family and functions like a molecular chaperone, and may thus help in maintaining the transparency of the eye lens by protecting the lens proteins from various stress conditions. Non-enzymic glycation of long-lived proteins has been implicated in several age- and diabetes-related complications, including cataract. Dicarbonyl compounds such as methylglyoxal and glyoxal have been identified as the predominant source for the formation of advanced glycation end-products in various tissues including the lens. We have investigated the effect of non-enzymic browning of alpha-crystallin by reactive dicarbonyls on its molecular chaperone-like function. Non-enzymic browning of bovine alpha-crystallin in vitro caused, along with altered secondary and tertiary structures, cross-linking and high-molecular-mass aggregation. Notwithstanding these structural changes, methylglyoxal- and glyoxal-modified alpha-crystallin showed enhanced anti-aggregation activity in various in vitro aggregation assays. Paradoxically, increased chaperone-like activity of modified alpha-crystallin was not associated with increased surface hydrophobicity and rather showed less 8-anilinonaphthalene-l-sulphonic acid binding. In contrast, the chaperone-like function of modified alpha-crystallin was found to be reduced in assays that monitor the prevention of enzyme inactivation by UV-B and heat. Moreover, incubation of bovine lens with methylglyoxal in organ culture resulted in cataract formation with accumulation of advanced glycation end-products and recovery of alpha-crystallin in high proportions in the insoluble fraction. Furthermore, soluble alpha-crystallin from methylglyoxal-treated lenses showed decreased chaperone-like activity. Thus, in addition to describing the effects of methylglyoxal and glyoxal on structure and chaperone-like activity, our studies also bring out an important caveat of aggregation assays in the context of the chaperone function of alpha-crystallin.