Qihao Zhang

Overview on the recent study of antimicrobial peptides: Origins, functions, relative mechanisms and application

Antimicrobial peptides (AMPs), which are produced by several species including insects, other animals, micro-organisms and synthesis, are a critical component of the natural defense system. With the growing problem of pathogenic organisms resistant to conventional antibiotics, especially with the emergence of NDM-1, there is increased interest in the pharmacological application of AMPs. They can protect against a broad array of infectious agents, such as bacteria, fungi, parasite, virus and cancer cells. AMPs have a very good future in the application in pharmaceuticals industry and food additive. This review focuses on the AMPs from different origins in these recent years, and discusses their various functions and relative mechanisms of action. It will provide some detailed files for clinical research of pharmaceuticals industry and food additive in application.

NGF induces adult stem Leydig cells to proliferate and differentiate during Leydig cell regeneration.

Nerve growth factor (NGF) has been reported to be involved in male reproductive physiology. However, few reports have described the activity of NGF during Leydig cell development. The objective of the present study was to examine the role of NGF during stem-Leydig-cell (SLC) regeneration. We investigated the effects of NGF on Leydig-cell (LC) regeneration by measuring mRNA levels in the adult rat testis after ethane dimethanesulfonate (EDS) treatment. Furthermore, we used the established organ culture model of rat seminiferous tubules to examine the regulation of NGF during SLC proliferation and differentiation using EdU staining, real-time PCR and western blotting. Progenitor Leydig cells (PLCs) and immature Leydig cells (ILCs) were also used to investigate the effects of NGF on LCs at different developmental stages. NGF mRNA levels changed significantly during Leydig-cell regeneration in vivo. In vitro, NGF significantly promoted the proliferation of stem Leydig cells and also induced steroidogenic enzyme gene expression and 3β-HSD protein expression. The data from PLCs and ILCs showed that NGF could increase Cyclin D1 and Hsd 17b3 mRNA levels in PLCs and Cyclin D1 mRNA levels in ILCs. These results indicate that NGF may play an important role during LC regeneration by regulating the proliferation and differentiation of LCs at different developmental stages, from SLCs to PLCs and from PLCs to ILCs. The discovery of this effect of NGF on Leydig cells will provide useful information for developing new potential therapies for PADAM (Partial Androgen Deficiency in the Aging Male).

Dihydrolipoamide dehydrogenase and cAMP are associated with cadmium-mediated Leydig cell damage.

Cadmium (Cd) directly inhibits testosterone production in Leydig cells, but its mechanism is still unclear. To further explore the signaling pathway of Cd-mediated toxicity to Leydig cells, various concentrations of Cd were cultured with R2C cells for 24h, and two-dimensional gel electrophoresis (2DE)-based proteomics profiling was used to analyze the change of protein expressions. Cd caused a concentration-dependent inhibition of cell viability with IC(25), IC(50) and IC(75) of 2.42×10(-5)M, 4.83×10(-5)M and 7.39×10(-5)M, respectively. Cd significantly reduced progesterone production and mitochondrial membrane potential (ΔΨ(m)) in a concentration-dependent manner. 2DE-based proteomics showed 34 protein spots with altered expression by 2-folds or more, and dihydrolipoamide dehydrogenase (DLD) was the hub in the network of these altered proteins. Real-time polymerase chain reaction (PCR) and Western blotting showed that Cd downregulated the expression of DLD. Cd also decreased intracellular levels of cyclic adenosine monophosphate (cAMP). The results suggest that DLD and cAMP may be key elements related to Cd toxicity to Leydig cells.

Intranasal administration of TAT-haFGF14–154 attenuates disease progression in a mouse model of Alzheimer’s disease

Human acidic fibroblast growth factor (haFGF), a neurotrophin-like growth factor in the brain, plays important roles in the development, differentiation and regeneration of brain neurons, which makes it potential to treat Alzheimers disease (AD). In this study, haFGF(14-154) and TAT-haFGF(14-154) (haFGF(14-154) fused with the cell-penetrating peptide transactivator of transcription protein transduction domain (TAT-PTD)) were intranasally administrated for 5 weeks to investigate the effects on senescence-accelerated mouse prone-8 (SAMP8) mice (a mouse model of AD). Results showed that TAT-PTD could increase the concentration of haFGF in the brain significantly, and TAT-haFGF(14-154) was more effective than haFGF(14-154) in the same dosage (300 μg/kg). Importantly, TAT-haFGF(14-154) improved the learning and memory abilities of SAMP8 mice in the behavioral test, and promoted the function of cholinergic system by measuring the relevant biomarkers (acetylcholine (ACh) level, acetylcholinesterase (AChE) and choline acetyltransferase (ChAT) activities). TAT-haFGF(14-154) also significantly reduced β-amyloid protein(1-42) (Aβ(1-42)) deposits as well as the levels of Aβ soluble forms in the mice brains and prevented the neurons from apoptosis. Besides, the oxidative stress impairment in the brain and serum was also ameliorated. The results suggest that TAT-haFGF(14-154) could attenuate the disease progression of SAMP8 AD mice, and the mechanism is related to the regulation of neurons microenvironment including neurotransmitters, Aβ pathology and oxidative stress.

Basic fibroblast growth factor promotes stem Leydig cell development and inhibits LH-stimulated androgen production by regulating microRNA expression.

Leydig cells are the primary source of testosterone in the testes, and their steroidogenic function is strictly controlled by the hypothalamus-pituitary-gonad axis. Emerging evidence has indicated that fibroblast growth factors play a role in regulating stem Leydig cell development and steroidogenesis, but little is known about the regulatory mechanism. Using a seminiferous tubule culture system, we demonstrated that basic fibroblast growth factor (bFGF) can promote stem Leydig cell proliferation and commitment toward differentiation in testosterone-producing Leydig cells. However, these promoting effects decreased with an increase in the bFGF dose. Previous studies have reported that bFGF inhibits luteinizing hormone (LH)-stimulated androgen production by downregulating the mRNA expression of steroidogenic genes in immature Leydig cells. However, the expression levels of 677 microRNAs did not change significantly during the LH-mediated process of testosterone synthesis. Five microRNAs (miR-29a, -29c, -142-3p, -451 and -335) were identified, and their expression in immature Leydig cells was regulated simultaneously by bFGF and LH. These results suggested that the inhibition of LH-stimulated androgen production may be modulated by a change in bFGF-mediated microRNA expression, which further impacts the signaling pathway of testosterone biosynthesis and steroidogenic gene expression.

Heavy metals chromium and neodymium reduced phosphorylation level of heat shock protein 27 in human keratinocytes

Heavy metals may exert their acute and chronic effects on the human skin through stress signals. In the present study, 2DE-based proteomics was used to analyze the protein expression in human keratinocytes exposed to heavy metals, chromium and neodymium, and 10 proteins with altered expression were identified. Among these proteins, small heat shock protein 27 (HSP27) was up-regulated significantly and the up-regulation was validated by Western blot and immunofluorescence. In addition, the mRNA expression level of HSP27 markedly increased as detected by quantitative PCR. More interestingly, the ratio of phosphorylated HSP27 and total HSP27 significantly decreased in keratinocytes treated with the heavy metals. These findings suggested that heavy metals reduced the phosphorylation level of HSP27, and that the ratio of p-HSP27 and HSP27 may represent a potential marker or additional endpoint for the hazard assessment of skin irritation caused by chemical products.

Toxic Mechanisms of 3-Monochloropropane-1,2-Diol on Progesterone Production in R2C Rat Leydig Cells

3-Monochloropropane-1,2-diol (3-MCPD) is a well-known food processing contaminant that has been shown to impede the male reproductive function. However, its mechanism of action remains to be elucidated. In this study, the effects of 3-MCPD on progesterone production were investigated using R2C Leydig cells. 3-MCPD caused concentration-dependent inhibition of cell viability at the IC25, IC50, and IC75 levels of 1.027, 1.802, and 3.160 mM, respectively. Single cell gel/comet assay and atomic force microscopy assay showed that 3-MCPD significantly induced early apoptosis. In addition, 3-MCPD significantly reduced progesterone production by reducing the expression of cytochrome P450 side-chain cleavage enzyme, steroidogenic acute regulatory protein, and 3β-hydroxysteroid dehydrogenase in R2C cells. The change in steroidogenic acute regulatory protein expression was highly consistent with progesterone production. Furthermore, the mitochondrial membrane potential and cAMP significantly decreased.

Identification of a novel aFGF-binding peptide with anti-tumor effect on breast cancer from phage display library

It has been reported that acidic fibroblast growth factor (aFGF) is expressed in breast cancer and via interactions with fibroblast growth factor receptors (FGFRs) to promote the stage and grade of the disease. Thus, aFGF/FGFRs have been considered essential targets in breast cancer therapy. We identified a specific aFGF-binding peptide (AGNWTPI, named AP8) from a phage display heptapeptide library with aFGF after four rounds of biopanning. The peptide AP8 contained two (TP) amino acids identical and showed high homology to the peptides of the 182-188 (GTPNPTL) site of high-affinity aFGF receptor FGFR1. Functional analyses indicated that AP8 specifically competed with the corresponding phage clone A8 for binding to aFGF. In addition, AP8 could inhibit aFGF-stimulated cell proliferation, arrested the cell cycle at the G0/G1 phase by increasing PA2G4 and suppressing Cyclin D1 and PCNA, and blocked the aFGF-induced activation of Erk1/2 and Akt kinase in both breast cancer cells and vascular endothelial cells. Therefore, these results indicate that peptide AP8, acting as an aFGF antagonist, is a promising therapeutic agent for the treatment of breast cancer.

Identification of potential biomarkers for predicting acute dermal irritation by proteomic analysis

In vitro alternative tests aiming at replacing the traditional animal test for predicting the irritant potential of chemicals have been developed, but the assessment parameters or endpoints are still not sufficient for analysis. To discover novel endpoints for skin irritation responses, a proteomics approach was used to analyze the protein expression in human keratinocytes exposed to sodium lauryl sulfate in the present study. Among the 20 identified proteins with altered expression, small heat shock protein 27 (HSP27) and superoxide dismutase [Cu–Zn] were down‐regulated while cofilin‐1 was up‐regulated significantly in response to the chemical challenge. Keratinocytes were exposed to acid and basic chemicals for further validation of the proteins. HSP27 displayed the most significant alteration both in mRNA and protein levels, accompanied by nuclear translocation. The irritation also induced an increased production of interleukin‐1α in keratinocytes. These findings suggest that these proteins may be combinational biomarkers or additional endpoints for skin hazard assessment. Further investigation into the protein alterations would be helpful for the mechanistic understanding of skin irritation. Copyright

Expression and Purification of Glutathione Transferase-Small Ubiquitin-Related Modifier-Metallothionein Fusion Protein and Its Neuronal and Hepatic Protection against d-Galactose-Induced Oxidative Damage in Mouse Model

The present study aimed to produce and pathophysiologically evaluate the metallothionein (MT) fusion protein. A recombinant plasmid containing DNA segment coding the pET-glutathione transferase (GST)-small ubiquitin-related modifier (SUMO)-MT fusion protein was inserted into Escherichia coli for expression. The expression level of the fusion protein was very high, reaching to 38.4% of the total supernatant proteins from the organism. Subsequent filtration through glutathione Sepharose 4B gel and Sephadex G-25 yielded an MT fusion protein with purity more than 95%. When exposed to metals, E. coli containing the GST-SUMO-MT fusion protein showed an increased accumulation of Cd2+, Zn2+, or Cu2+ at approximately 4.2, 4.0, or 1.6 times higher, respectively, than those containing the control protein. Administration of GST-SUMO-MT to mice that were also treated with d-galactose to induce neuronal and hepatic damage showed a significant improvement of animal learning and memory capacity, which was depressed in mice treated by d-galactose alone. Administration of MT fusion protein also prevented d-galactose-increased malondialdehyde contents and histopathological changes in the brain and liver. Furthermore, supplement of the fusion protein significantly prevented d-galactose-increased nitric oxide contents and -decreased superoxide dismutase activity in the brain, liver, and serum. The fusion protein was also able to prevent ionizing radiation-induced DNA damage of the mouse thymus. The present study indicates that GST-SUMO-MT has a normal metal binding feature and also significantly protects the multiple tissues against oxidative damage in vivo caused by chronic exposure to d-galactose and by ionizing radiation. Therefore, GST-SUMO-MT may be a potential candidate to be developed for the clinical application.