Excessive zinc in diet induces leptin resistance in Wistar rat through increased uptake of nutrients at intestinal level

ProjectThe ob gene has either been found to be mutant defective resulting in a deficiency of its product leptin or leptin has been found to be resistant to its receptors in obese human and rodents. The factors inducing leptin resistance have not been identified. Since excessive bioavailability of Zn has been implicated in obesity, we investigated if its excess in diet induces leptin resistance.ProcedureFor the investigations, three groups of Wistar rats were included in this study and they were fed on equicalories semi synthetic basal diet containing 20 mg, 40 mg or 80 mg Zn/kg diet for 120 days. There after they were sacrificed for hormonal status and intestinal investigations.ResultsThe data of this study revealed that the food intake, gain in body weight, serum leptin, glucose, insulin, cortisol increased with increased Zn concentration in diet. TEM study showed a positive correlation between Zn concentration in diet and number of microvilli/unit surface area of the mucosal epithelial cells of the intestine.ConclusionThe results of this study suggest that excessive bioavailability of Zn induces leptin resistance through increased uptake of nutrients at intestinal level, leading to the growth of the fat cells which aggravated the leptin synthesis and its release in the blood stream. In spite of its higher circulating level, it was unable to reduce the food intake and gain in body weight in Zn treated rats equivalent to the control group.     

Environmental and epidemiological surveillance of Vibrio cholerae in a cholera-endemic region in India with freshwater environs

Aim: To conduct epidemiological and ecological surveillance of cholera in freshwater environments. Methods and Results: A freshwater region of India was surveyed between April 2007 and December 2008. Vibrio cholerae was isolated from 59·5% of water and plankton samples (n = 357) and 35·5% of stool samples (n = 290). Isolation from water was dependent on air (r = 0·44) and water temperatures (r = 0·49) (P < 0·01) but was independent of rainfall (r = 0·15), chlorophyll a (r = 0·18), salinity (r = 0·2) or pH (r = 0·2) (P > 0·05). Isolation from plankton was dependent on temperature of air (r = 0·45), water temperature (r = 0·44), chlorophyll a concentration (r = 0·42), pH (r = 0·23) and salinity (r = 0·39) (P < 0·01). Cholera cases correlated with rainfall (r = 0·82, P < 0·01) and chlorophyll a concentration (r = 0·42, P < 0·05), but not with air temperature (r = 0·3, P = 0·37). Vibrio cholerae O1 possessed ctxB, ctxA, rstR and tcpA (ElTor), toxR, toxT, rtxA, rtxC, mshA and hylA. Among non‐O1–non‐O139, the distribution of virulence‐associated and regulatory protein genes was heterogeneous with – 0·7, 2·2, 94·77, 97·76, 99·25, 100 and 100% isolates being positive for tcpA, toxT, rtxA, rtxC, hylA, toxR and mshA, respectively. Two‐thirds of non‐O1–non‐O139 isolates exhibited antibiotic resistance to various antibiotics that did not correlate with geographical site or time of origin for the isolates. RAPD and AFLP showed V. cholerae to be a diverse bacterium. AFLP demonstrated separate lineages for non‐O1–non‐O139 and O1 isolates. Conclusion: Environmental parameters played a significant role in the emergence and spread of cholera and the abundance of V. cholerae. But based on virulence gene profiling and genetic fingerprinting, the possibility of origin of toxigenic isolates from nontoxigenic environmental isolates seems unlikely in freshwater environs of India. Significance and Impact of the Study: This study explains the ecology, epidemiology and seasonality of cholera in freshwater environs.     

Delineating the role of the HLA-DR4 "shared epitope" in susceptibility versus resistance to develop arthritis

In humans, HLA-DR alleles sharing amino acids at the third hypervariable region with DRB1*0401(shared epitope) are associated with a predisposition to rheumatoid arthritis, whereas DRB1*0402 is not associated with such a predisposition. Both DRB1*0402 and DRB1*0401 occur in linkage with DQ8 (DQB1*0302). We have previously shown that transgenic (Tg) mice expressing HLA-DRB1*0401 develop collagen-induced arthritis. To delineate the role of "shared epitope" and gene complementation between DR and DQ in arthritis, we generated DRB1*0402, DRB1*0401.DQ8, and DRB1*0402.DQ8 Tg mice lacking endogenous class II molecules, AE(o). DRB1*0402 mice are resistant to develop arthritis. In double-Tg mice, the DRB1*0401 gene contributes to the development of collagen-induced arthritis, whereas DRB1*0402 prevents the disease. Humoral response to type II collagen is not defective in resistant mice, although cellular response to type II collagen is lower in *0402 mice compared with *0401 mice. *0402 mice have lower numbers of T cells in thymus compared with *0401 mice, suggesting that the protective effect could be due to deletion of autoreactive T cells. Additionally, DRB1*0402 mice have a higher number of regulatory T cells and show increased activation-induced cell death, which might contribute toward protection. In DRB1*0401.DQ8 mice, activated CD4(+) T cells express class II genes and can present DR4- and DQ8-restricted peptides in vitro, suggesting a role of class II(+) CD4 T cells locally in the joints. The data suggest that polymorphism in DRB1 genes determines predisposition to develop arthritis by shaping the T cell repertoire in thymus and activating autoreactive or regulatory T cells.     

Deregulated Cdk5 promotes oxidative stress and mitochondrial dysfunction

Oxidative stress is one of the earliest events in Alzheimer's disease (AD). A chemical genetic screen revealed that deregulated cyclin-dependent kinase 5 (Cdk5) may cause oxidative stress by compromising the cellular anti-oxidant defense system. Using novel Cdk5 modulators, we show the mechanism by which Cdk5 can induce oxidative stress in the disease's early stage and cell death in the late stage. Cdk5 dysregulation upon neurotoxic insults results in reactive oxygen species (ROS) accumulation in neuronal cells because of the inactivation of peroxiredoxin I and II. Sole temporal activation of Cdk5 also increases ROS, suggesting its major role in this process. Cdk5 inhibition rescues mitochondrial damage upon neurotoxic insults, thereby revealing Cdk5 as an upstream regulator of mitochondrial dysfunction. As mitochondrial damage results in elevated ROS and Ca(2+) levels, both of which activate Cdk5, we propose that a feedback loop occurs in late stage of AD and leads to cell death (active Cdk5 --> ROS --> excess ROS --> mitochondrial damage --> ROS --> hyperactive Cdk5 --> severe oxidative stress and cell injury --> cell death). Cdk5 inhibition upon neurotoxic insult prevents cell death significantly, supporting this hypothesis. As oxidative stress and mitochondrial dysfunction play pivotal roles in promoting neurodegeneration, Cdk5 could be a viable therapeutic target for AD.     

Redundancy and plasticity of neutralizing antibody responses are cornerstone attributes of the human immune response to the smallpox vaccine

The smallpox vaccine is widely considered the gold standard for human vaccines, yet the key antibody targets in humans remain unclear. We endeavored to identify a stereotypic, dominant, mature virion (MV) neutralizing antibody target in humans which could be used as a diagnostic serological marker of protective humoral immunity induced by the smallpox vaccine (vaccinia virus [VACV]). We have instead found that diversity is a defining characteristic of the human antibody response to the smallpox vaccine. We show that H3 is the most immunodominant VACV neutralizing antibody target, as determined by correlation analysis of immunoglobulin G (IgG) specificities to MV neutralizing antibody titers. It was determined that purified human anti-H3 IgG is sufficient for neutralization of VACV; however, depletion or blockade of anti-H3 antibodies revealed no significant reduction in neutralization activity, showing anti-H3 IgG is not required in vaccinated humans (or mice) for neutralization of MV. Comparable results were obtained for human (and mouse) anti-L1 IgG and even for anti-H3 and anti-L1 IgG in combination. In addition to H3 and L1, human antibody responses to D8, A27, D13, and A14 exhibited statistically significant correlations with virus neutralization. Altogether, these data indicate the smallpox vaccine succeeds in generating strong neutralizing antibody responses not by eliciting a stereotypic response to a single key antigen but instead by driving development of neutralizing antibodies to multiple viral proteins, resulting in a "safety net" of highly redundant neutralizing antibody responses, the specificities of which can vary from individual to individual. We propose that this is a fundamental attribute of the smallpox vaccine.     

Attenuation of Zn-Induced Acute Pancreatitis in Wistar Rat Fed on Cu- and Mg- Enriched Modified Poultry Egg^{\Psi }

Zinc (Zn) consumption has increased in many populations either due to the increased intake of Zn-fortified foods as in the USA or in agricultural food stuffs as in some Indian states during the last decade. Its excessive intake has been reported to induce acute pancreatitis (AP) in many studies due to increase in oxidative stress that was further reported to cause Cu and Mg deficiencies. This led us to design a modified poultry egg (ME^Ψ) enriched with Cu and Mg along with other antioxidants, and its efficacy on Zn-induced AP was studied in male Wistar rats. In one set, the rats were fed on equacaloric semi-synthetic basal diet containing 20 mg Zn/kg diet (control, group I), and Zn-induced AP-I diet and AP-II diet containing 40 and 80 mg Zn/kg diet (groups II and III) for 180 days, respectively. In another set, the rats were initially fed on Zn-induced AP-I and AP-II diets for 90 days and then shifted to -mixed Zn-induced AP-I and AP-II diets in groups IIME and IIIME for another 90 days. At the end of the experiment, data displayed increased serum and urinary Zn, Cu, and Mg levels in groups II and III rats, which were reduced and approached closer to control group I after feeding in groups IIME and IIIME rats. Transmission electron microscopic studies of acinar cells revealed progressive dilation, vesicularization, and degeneration of endoplasmic reticulum (ER), and decrease in zymogen granules (ZG) in groups II and III rats in contrast to their curvilinear or concentric long parallel running cisternal profile of ER in control group I. The treatment of helped in the restoration of the ER profile and ZG number, approaching closer to the control group I. The degree of recovery was dependent upon the degree of toxicity caused by the amount of Zn given in the diet. The results of this study suggest that -mixed diet can protect the acinar cells from the deleterious effects of Zn by decreasing the oxidative stress. Indian patent application no. 2264/Del/2005.     

Assessment of Minerals in Obesity-related Diseases in the Chandigarh (India) Population

Excessive Zn but normal Cu and Mg in the staple food consumed by the people of Chandigarh (Union territory and capital of Punjab and Haryana States of India) has been considered to be the major risk factor for the prevalence of obesity (33.15%) and obesity-related diseases in this region. Therefore, in the present investigations, in obesity-related diseases, the status of these minerals was estimated in their tissues, including hair, nails, and blood serum and urine, and compared with those of normal subjects. They were grouped as: normal subjects in control Group A, middle-aged diabetics in Group D_M, older diabetics in Group D_O, and diabetics with osteoarthritis in Group D+ OA, osteoarthritis in Group OA and rheumatoid arthritis in Group RA, respectively. The results evaluated in the order as: hair Zn, group D+OA>D_M>OA>A (control)>RA>D_O (p < 0.001); hair Cu, group A (control)>D_M>OA>D+OA>D_O>RA (p < 0.001); hair Mg, group A (control)>D_M>OA>D+OA>RA>D_O (p < 0.001, 0.01); hair Mn, group A (control)>RA>OA>D-OA>D_M>D_O (p < 0.001); nail Zn, group D_M>D+OA>OA>A (control)>RA>D_O (p < 0.001, 0.05); nail Cu, group A (control)>OA>D_M>D+OA>RA>D_O (p < 0.001); nail Mg, group A (control)>OA>D_M>D_O>D+OA >RA (p < 0.001); nail Mn, group A (control) >RA>OA>D+OA>D_M>D_O (p < 0.01); urine Zn, group D_O>D_M>D+OA>A (control)>RA>OA (p < 0.01); urine Cu, group RA>D+OA>D_O>OA> D_M>A (control) (p<0.001); urine Mg, group RA>OA>D+OA>D_O>D_M>A (control; p < 0.001); urine Mn, group D_O>D_M>OA>D+OA>RA>A (control; p < 0.001), respectively. The analysis of the mineral status in serum of diabetics further showed their highly significant rise from lower mean age subgroup to higher mean age subgroup than their control counter parts (p < 0.001, 0.01, and 0.05) with coincident deficiencies of Cu, Mg, and Mn in their tissues. This study would be helpful considering the status of minerals in these obesity-related diseases depending on the choice of the food consumed to improve the quality of life and prognosis for the diseases.     

An essential oil and its major constituent isointermedeol induce apoptosis by increased expression of mitochondrial cytochrome c and apical death receptors in human leukaemia HL-60 cells

An essential oil from a lemon grass variety of Cymbopogon flexuosus (CFO) and its major chemical constituent sesquiterpene isointermedeol (ISO) were investigated for their ability to induce apoptosis in human leukaemia HL-60 cells because dysregulation of apoptosis is the hallmark of cancer cells. CFO and ISO inhibited cell proliferation with 48 h IC50 of approximately 30 and 20 microg/ml, respectively. Both induced concentration dependent strong and early apoptosis as measured by various end-points, e.g. annexinV binding, DNA laddering, apoptotic bodies formation and an increase in hypo diploid sub-G0 DNA content during the early 6h period of study. This could be because of early surge in ROS formation with concurrent loss of mitochondrial membrane potential observed. Both CFO and ISO activated apical death receptors TNFR1, DR4 and caspase-8 activity. Simultaneously, both increased the expression of mitochondrial cytochrome c protein with its concomitant release to cytosol leading to caspase-9 activation, suggesting thereby the involvement of both the intrinsic and extrinsic pathways of apoptosis. Further, Bax translocation, and decrease in nuclear NF-kappaB expression predict multi-target effects of the essential oil and ISO while both appeared to follow similar signaling apoptosis pathways. The easy and abundant availability of the oil combined with its suggested mechanism of cytotoxicity make CFO highly useful in the development of anti-cancer therapeutics.