Zhongxin Zhu

Metallothionein prevents cardiac pathological changes in diabetes by modulating nitration and inactivation of cardiac ATP synthase.

Mitochondrial ATP production is the main energy source for the cell. Diabetes reduces the efficient generation of ATP, possibly due to the inactivation of ATP synthase. However, the exact mechanism by which diabetes induces inactivation of ATP synthase remains unknown, as well as whether such inactivation has a role in the development of pathological abnormalities of the diabetic heart. To address these issues, we used cardiac metallothionein-transgenic (MT-TG) and wild-type (WT) mice with streptozotocin-induced diabetes, since we have demonstrated previously that diabetes-induced cardiac damage and remodeling were found in WT diabetic mice, but not in MT-TG diabetic mice. Immunohistochemical and biochemical assays were used to compare pathological and biochemical changes of the heart between MT-TG and WT diabetic mice, and a proteomic assay to evaluate ATP synthase expression and tyrosine nitration, with its activity. LC/MS analysis revealed that diabetes increased tyrosine nitration of the ATP synthase α subunit at Tyr(271), Tyr(311), and Tyr(476), and the β subunit at Tyr(269) and Tyr(508), and also significantly reduced ATP synthase activity by ~32%. These changes were not observed in MT-TG diabetic mice. Furthermore, parallel experiments with induced expression of cardiac MT by zinc supplementation in diabetic mice produced similar effects. These results suggest that MT can preserve ATP synthase activity in streptozotocin-induced diabetes, probably through the inhibition of ATP synthase nitration.

An improved silver stain for the visualization of lipopolysaccharides on polyacrylamide gels.

A sensitive, brief, and user‐friendly silver stain to meet the needs in high‐efficiency detection of lipopolysaccharides (LPS) on polyacrylamide gels is described. In this study, the most commonly used formaldehyde‐based LPS silver stain, which is potentially hazardous to the operator, is replaced by ascorbic acid (Vc) in alkaline sodium thiosulfate solution. It takes only about 35 min to complete all the protocol, with a detection limit of 4 ng of total LPS. The results indicate that this user‐friendly method could be a good choice for LPS visualization on polyacrylamide gels.

The activation of the NF-κB-JNK pathway is independent of the PI3K-Rac1-JNK pathway involved in the bFGF-regulated human fibroblast cell migration

BACKGROUND Skin wound healing is a complex process that repairs multiple organ-tissues. Fibroblasts are key players of skin cells, whose migration is important during wound healing process. bFGF has shown a great efficacy to promote cell migration, but the precise mechanism by which bFGF regulates cell migration remains elusive. OBJECTIVE The aim of this study was to find bFGF-regulated gene pools and further identify target molecules that participated in human fibroblast cell migration. METHODS Skin primary fibroblasts and rat skin wound model were used to demonstrate the novel mechanism of bFGF regulating cell migration to accelerate wound healing. Cell migration was determined using the wound healing scratch assay. The differentially expressed genes and numerous biochemical pathways after bFGF treatment were identified by RNA-Seq analysis, and differentially expressed genes were further verified by qRT-PCR. siRNA duplex target to interfering the expression of PI3-kinase (p110α) was transformed into NIH/3T3 cells. Western blotting analysis was used to determine marker protein expressions. The invasive activity of fibroblasts was measured using 3D spheroid cell invasion assay. RESULTS RNA-Seq analysis identified numerous biochemical pathways including the NF-κB pathway under the control of FGF signaling. bFGF negatively regulates the phosphorylation of IκB-α, the most well studied NF-κB signaling regulator while bFGF induces JNK phosphorylation. Application of Bay11-7082, a representative NF-κB inhibitor promoted cell migration, invasion and enhanced the JNKs phosphorylation. However, inhibition of JNKs blocked cell migration when NF-κB is inhibited. Moreover, application of the PI3K inhibitor LY294002 together with Bay11-7082 maintained normal cell migration and knocking-down PI3K (p110α) by a specific siRNA inhibited JNKs phosphorylation while maintaining normal IκBα phosphorylation, indicating that PI3K and NF-κB signaling independently regulate JNKs activation. In addition, administration of bFGF or Bay11-7082 promoted rat skin wound repair and accelerated the invasion of fibroblasts. CONCLUSION This study sheds light on the mode of action of bFGF and identifies that the NF-κB-JNKs pathway is independent of the PI3K-JNKs pathway to accelerate fibroblast migration. In addition, bFGF and the relief of inflammation could be a favorable therapeutic approach for skin wound healing.

Highly Sensitive Method for Specific, Brief, and Economical Detection of Glycoproteins in Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis by the Synthesis of a New Hydrazide Derivative

A new hydrazide derivative was synthesized and used for the first time as a specific, brief, and economical probe to selectively visualize glycoproteins in 1-D and 2-D sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with high sensitivity. The detection limit of the newly developed staining method is 2- and 4-fold higher than that of the widely used Pro-Q Emerald 300 and 488 stains, respectively.

Metallothionein prevents diabetes-induced cardiac pathological changes, likely via the inhibition of succinyl-CoA:3-ketoacid coenzyme A transferase-1 nitration at Trp374

We previously demonstrated that metallothionein (MT)-mediated protection from diabetes-induced pathological changes in cardiac tissues is related to suppression of superoxide generation and protein nitration. The present study investigated which diabetes-nitrated protein(s) mediate the development of these pathological changes by identifying the panel of nitrated proteins present in diabetic hearts of wild-type (WT) mice and not in those of cardiac-specific MT-overexpressing transgenic (MT-TG) mice. At 2, 4, 8, and 16 wk after streptozotocin induction of diabetes, histopathological examination of the WT and MT-TG diabetic hearts revealed cardiac structure derangement and remodeling, significantly increased superoxide generation, and 3-nitrotyrosine accumulation. A nitrated protein of 58 kDa, succinyl-CoA:3-ketoacid CoA transferase-1 (SCOT), was identified by mass spectrometry. Although total SCOT expression was not significantly different between the two types of mice, the diabetic WT hearts showed significantly increased nitration content and dramatically decreased catalyzing activity of SCOT. Although SCOT nitration sites were identified at Tyr(76), Tyr(117), Tyr(135), Tyr(226), Tyr(368), and Trp(374), only Tyr(76) and Trp(374) were found to be located in the active site by three-dimensional structure modeling. However, only Trp(374) showed a significantly different nitration level between the WT and MT-TG diabetic hearts. These results suggest that MT prevention of diabetes-induced pathological changes in cardiac tissues is most likely mediated by suppression of SCOT nitration at Trp(374).

Acute ethanol exposure‐induced autophagy‐mediated cardiac injury via activation of the ROS‐JNK‐Bcl‐2 pathway

Binge drinking is associated with increased cardiac autophagy, and often triggers heart injury. Given the essential role of autophagy in various cardiac diseases, this study was designed to investigate the role of autophagy in ethanol‐induced cardiac injury and the underlying mechanism. Our study showed that ethanol exposure enhanced the levels of LC3‐II and LC3‐II positive puncta and promoted cardiomyocyte apoptosis in vivo and in vitro. In addition, we found that ethanol induced autophagy and cardiac injury largely via the sequential triggering of reactive oxygen species (ROS) accumulation, activation of c‐Jun NH2‐terminal kinase (JNK), phosphorylation of Bcl‐2, and dissociation of the Beclin 1/Bcl‐2 complex. By contrast, inhibition of ethanol‐induced autophagic flux with pharmacologic agents in the hearts of mice and cultured cells significantly alleviated ethanol‐induced cardiomyocyte apoptosis and heart injury. Elimination of ROS with the antioxidant N‐acetyl cysteine (NAC) or inhibition of JNK with the JNK inhibitor SP600125 reduced ethanol‐induced autophagy and subsequent autophagy‐mediated apoptosis. Moreover, metallothionein (MT), which can scavenge reactive oxygen and nitrogen species, also attenuated ethanol‐induced autophagy and cell apoptosis in MT‐TG mice. In conclusion, our findings suggest that acute ethanol exposure induced autophagy‐mediated heart toxicity and injury mainly through the ROS‐JNK‐Bcl‐2 signaling pathway.

An expanding negative detection method for the visualization of DNA in polyacrylamide gels by eosin Y

A sensitive and easy technique has been developed for the negative detection of DNA following PAGE using eosin Y. After electrophoresis, gels are fixed and stained within 40 min to provide a detection limit of 0.1–0.2 ng of single DNA band, which appears as transparent and colorless under the opaque gel matrix background. The sensitivity of the new stain is fourfold better than zinc‐imidazole negative and ethidium bromide stains. Furthermore, the newly developed staining method has been successfully applied to RNA visualization in polyacrylamide gels. In addition, the inclusion of inorganic salts in staining solution was also investigated, which has great effect on the staining efficiency.

A visible dye-based staining method for DNA in polyacrylamide gels by ethyl violet

We describe a visible dye-based staining method for DNA in polyacrylamide gels using ethyl violet (EV). The novel method is a background-free, sensitive, economical, and simple procedure involving only staining and washing steps that can be completed within 30 min. As little as 0.8-1.6 ng of phiX174 DNA/HaeIII can be detected by EV, which is about eightfold more sensitive than Nile blue (NB) stain and twofold less sensitive than ethidium bromide (EB) stain.

Negative staining of lipopolysaccharides on polyacrylamide gels by using eosin B

A sensitive and simple technique for the negative detection of lipopolysaccharides (LPSs) following polyacrylamide gel electrophoresis (PAGE) using eosin B (EB) was developed. After electrophoresis, gels were fixed, stained, and developed within 30 min to achieve transparent and colorless LPS bands under opaque gel matrix background. As low as 20 to 40 ng of total LPSs could be detected, which is 4-fold more sensitive than those of the widely used silver stain developed by Fomsgaard and coworkers and imidazole-zinc (IZ) negative stain. For its sensitivity and brevity, this stain may be a practical method for LPS determination in the routine laboratory.

Improved staining of phosphoproteins with high sensitivity in polyacrylamide gels using Stains-All

An improved Stains‐All (ISA) staining method for phosphoproteins in SDS‐PAGE was described. Down to 0.5–1 ng phosphoproteins (α‐casein, β‐casein, or phosvitin) can be successfully selectively detected by ISA stain, which is approximately 120‐fold higher than that of original Stains‐All stain, but is similar to that of commonly used Pro‐Q Diamond stain. Furthermore, unlike the original Stains‐All protocol that was time consuming and light unstable, ISA stain could be completed within 60 min without resorting to protect the gels from light during the whole staining procedure. According to the results, it is concluded that ISA stain is a rapid, sensitive, specific, and economic staining method for a broad application to the research of phosphoproteins.