Yunbiao Wang

Understanding the mechanism of the dormant dauer formation of C. elegans: from genetics to biochemistry.

Dauer is a dormancy state that may occur at the end of developmental stage L1 or L2 of Caenorhabditis elegans when the environmental conditions are unfavorable (e.g., lack of food, high temperature, or overcrowding) for further growth. Dauer is a nonaging duration that does not affect the postdauer adult lifespan. Major molecular events would include the sensing of the environmental cues, the transduction of the signals into the cells, and the subsequent integration of the signals that result in the corresponding alteration of the metabolism and morphology of the organism. Genetics approach has been effectively used in identifying many of the so‐called daf genes involved in dauer formation using C. elegans as the model. Nevertheless, biochemical studies at the protein and metabolic level has been lacking behind in understanding this important life phenomenon. This review focuses on the biochemical understanding so far achieved on dauer formation and dormancy in general, as well as important issues that need to be addressed in the future.

Combined effect of temperature and zinc on Caenorhabditis elegans wild type and daf-21 mutant strains

Heavy metal pollution in aquatic ecosystems is a far reaching environmental problem. The possible influences of heavy metal exposure and the potential harm to organisms when combined with other environmental stressors such as temperature have been largely unexplored. An aquatic toxicity test of Caenorhabditis elegans was performed to estimate the 24h median lethal concentration (LC50) of different zinc concentrations at different temperatures (15°C, 20°C, 25°C, and 30°C). We also examined the time course thermotolerance on wild type (N2) and daf-21 null (JT6130) adults exposed to 6.1mM zinc at 37°C. Hsp90 protein expression level in response to the combined effect of temperature and zinc toxicity was also investigated by both Western blots and ELISA. Our results show that C. elegans wild type nematodes exhibit severe lethal toxicity after a 24h exposure to zinc at higher temperatures. In addition, the expression level of Hsp90 was highly inhibited in adult worms subjected to zinc stress. This toxicity assay at different temperatures provides insight into organism response to combined effects of temperature and zinc toxicity.

Regulation of lead toxicity by heat shock protein 90 (daf-21) is affected by temperature in Caenorhabditis elegans.

In the nematode Caenorhabditis elegans, stress resistance can be regulated by dauer formation (daf) genes. In the present study, regulation of heavy metal lead (Pb) toxicity by the 90-kDa heat shock proteins (Hsp90; daf-21) was investigated in both wild-type C. elegans and daf-21/Hsp90 mutants by focusing on the effects of varied temperatures below (15°C) or above (25 and 30°C) the presumptive optimum growth temperature (20°C). More acute toxicity of Pb, indicated by the 24-h median lethal concentrations (LC50), was observed in wild-type adults than in the daf-21 mutant adults at 15, 20 and 25°C; however, the daf-21 mutant adults showed more sensitivity at 30°C. Enhanced Pb sensitivity (e.g., decrease LC50) in both types of C. elegans was observed with both increased and decreased temperatures when compared to that at 20°C. Additional examined endpoints included time course of toxicity at LC50s, pharyngeal pumping, reproduction, life span, and Hsp90 expression. Collective results showed that temperatures both above and below 20°C exacerbated Pb toxicity, and that the protein level of daf-21/Hsp90 was one of the most sensitive indicators of Pb toxicity in wild-type C. elegans, while pharyngeal pumping was more Pb sensitive in daf-21 mutants. Therefore, the expression of daf-21/Hsp90 has apparent utility for the prediction and assessment of Pb-induced toxicity in nematodes. Further, the stress responses related to Hsp90 expression in C. elegans may have considerable potential as sensitive biomarkers for the monitoring of environmental Pb contamination.

Genistein modulation of streptozotocin diabetes in male B6C3F1 mice can be induced by diet

Diet and phytoestrogens affect the development and progression of diabetes. The objective of the present study was to determine if oral exposure to phytoestrogen genistein (GE) by gavage changed blood glucose levels (BGL) through immunomodulation in streptozotocin (STZ)-induced diabetic male B6C3F1 mice fed with three different diets. These three diets were: NTP-2000 diet (NTP), soy- and alfalfa-free 5K96 diet (SOF) and high fat diet (HFD) with 60% of kcal from fat, primarily rendered fat of swine. The dosing regimen for STZ consisted of three 100mg/kg doses (i.p.): the first dose was administered at approximately 2weeks following the initiation of daily GE (20mg/kg) gavage, and the second dose was on day 19 following the first dose, and the third dose was on day 57 following the first dose. In mice on the NTP diet, GE treatment decreased BGL with statistical significances observed on days 33 and 82 following the first STZ injection. In mice fed the HFD diet, GE treatment produced a significant decrease and a significant increase in BGL on days 15 and 89 following the first STZ injection, respectively. In mice fed the SOF diet, GE treatment had no significant effects on BGL. Although GE treatment affected phenotypic distributions of both splenocytes (T cells, B cells, natural killer cells and neutrophils) and thymocytes (CD4/CD8 and CD44/CD25), and their mitochondrial transmembrane potential and generation of reactive oxygen species, indicators of cell death (possibly apoptosis), GE modulation of neutrophils was more consistent with its diabetogenic or anti-diabetic potentials. The differential effects of GE on BGL in male B6C3F1 mice fed with three different diets with varied phytoestrogen contents suggest that the estrogenic properties of this compound may contribute to its modulation of diabetes.

Joint Toxicity of Arsenic, Copper and Glyphosate on Behavior, Reproduction and Heat Shock Protein Response in Caenorhabditis elegans

The soil nematode Caenorhabditis elegans was used in 24-h acute exposures to arsenic (As), copper (Cu) and glyphosate (GPS) and to mixtures of As/Cu and As/GPS to investigate the effects of mixture exposures in the worms. A synergistic type of interaction was observed for acute toxicity with the As/Cu and As/GPS mixtures. Sublethal 24-h exposures of 1/1000, 1/100 and 1/10 of the LC50 concentrations for As, Cu and GPS individually and for As/Cu and As/GPS mixtures were conducted to observe responses in locomotory behavior (head thrashing), reproduction, and heat shock protein expression. Head thrash frequency and reproduction exhibited concentration dependent decreases in both individual and combined exposures to the tested chemical stressors, and showed synergistic interactions even at micromolar concentrations. Furthermore, the HSP70 protein level was significantly increased following exposure to individual and combined chemical stressors in wild-type C. elegans. Our findings establish for the first time the effects of exposure to As/GPS and As/Cu mixtures in C. elegans.

Studying cytotoxicity of low concentration arsenic on PC 12 cell line

It is important to study toxicological effects of arsenic (As) on growth and function of mammal cell line because As is a well-known toxic and carcinogenic agent throughout the world. Herein, PC 12 cell line (rat pheochromo-cytoma cell line) has been selected as a mode cell line for evaluating toxicological effects of sodium arsenite (NaAsO2, As(III)) on nerve cells. The value (47 μM) of 24 h median lethal concentration (LC50) of NaAsO2 for PC 12 cells has been obtained by MTT assay. The cytotoxicity of NaAsO2 has been systematically studied by exposure of PC 12 cells to relatively low concentration (5 and 20 μM) of NaAsO2. We found that NaAsO2 is able to significantly decrease cell proliferation, generate strong genotoxicity and increase ROS (reactive oxygen species) level of PC 12 cells. The experimental results demonstrate that low concentration of (e.g., 5 μM, about 10% of its LC50) NaAsO2 can generate high cytotoxicity towards PC 12 cell line.

Heterologous expression of AgsA enhances Escherichia coli tolerance to the combined effect of elevated temperature and Zinc toxicity

Small heat shock proteins (sHSPs) are ubiquitous stress proteins that are able to protect the cells against cellular insults from temperature, heavy metal etc. However, the molecular chaperone roles of sHSPs in enhancing growth and adaptation under combined temperature and metal stresses in Escherichia coli cells have been poorly understood. Here, we analyze the function of recombinant AgsA, a small heat shock protein from Salmonella enterica serovar Typhimurium under combined temperature and zinc stress in E. coli. Our results show that the heterologous expression of AgsA significantly increases the tolerance of E. coli cells to the combined effect of temperature stress and zinc toxicity by maintaining the stability of soluble proteins. Furthermore, there was remarkable and significant difference in the half effect concentration (EC50) of zinc at all temperatures treatments in both test cells. The EC50s of zinc at 37 °C, 42 °C and 50 °C were 15.24 mg/L, 29.30 mg/L, and 5.98 mg/L respectively in the AgsA-transformed E. coli cells, and 3.03 mg/L, 2.38 mg/L, and 0.373 mg/L, respectively in the control cells lacking AgsA. Together, our data indicate a good concentration-response relationship between all three temperatures treatment and zinc toxicity in E. coli, and establish for the first time the combined effects of temperature and zinc toxicity on E. coli cells. Also, the AgsA protein response to combined thermal and metal stress could serve as a molecular biomarker for the assessment of interactive stress damage to the cells.