Hydroxysafflor yellow A (HYSA) inhibited the proliferation and differentiation of 3T3-L1 preadipocytes

Hydroxysafflor yellow A (HSYA), a main component of safflor yellow, has been demonstrated to prevent steroid-induced avascular necrosis of femoral head by inhibiting primary bone marrow-derived mesenchymal stromal cells adipogenic differentiation induced by steroid. In this study, we investigate the effect of HSYA on the proliferation and adipogenesis of mouse 3T3-L1 preadipocytes. The effects of HSYA on proliferation and differentiation of 3T3-L1 cells and its possible mechanism were studied by 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyl tetrazolium bromide spectrophotometry, Oil Red O staining, intracellular triglyceride assays, real-time quantitative RT-PCR, transient transfection and dual luciferase reporter gene methods. HSYA inhibited the proliferation of 3T3-L1 preadipocytes and cell viability greatly decreased in a dose and time dependent manner. HSYA (1 mg/l) notably reduced the amount of intracellular lipid and triglyceride content in adipocytes by 21.3 % (2.13 ± 0.36 vs 2.71 ± 0.40, P < 0.01) and 22.6 % (1.33 ± 0.07 vs 1.72 ± 0.07, P < 0.01) on days 8 following the differentiation, respectively. HSYA (1 mg/l) significantly increased hormone-sensitive lipase (HSL) mRNA expression and promoter activities by 2.4- and 1.55-fold, respectively (P < 0.01), in differentiated 3T3-L1 adipocytes. HSYA inhibits the proliferation and adipogenesis of 3T3-L1 preadipocytes. The inhibitory action of HYSA on adipogenesis may be due to the promotion of lipolytic-specific enzyme HSL expression by increasing HSL promoter activity.     

Granulocyte-macrophage colony-stimulating factor is a keratinocyte-derived factor involved in regulating the proliferation and differentiation of neonatal mouse epidermal melanocytes in culture

Mouse epidermal melanoblasts and melanocytes preferentially proliferated from disaggregated epidermal cell suspensions derived from newborn mouse skin in a serum-free melanocyte-proliferation medium (MDMD) and a melanoblast-proliferation medium (MDMDF) supplemented with dibutyryl adenosine 3':5'-cyclic monophosphate (DBcAMP) and/or basic fibroblast growth factor (bFGF). Pure cultured primary melanoblasts and melanocytes were further cultured with MDMD/MDMDF supplemented with granulocyte-macrophage colony-stimulating factor (GMCSF) from 14 days (keratinocyte depletion). GMCSF stimulated the number of melanoblasts/melanocytes as well as the percentage of differentiated melanocytes in keratinocyte-depleted cultures. Flow cytometry analysis showed that melanoblasts and melanocytes in the S and G(2)/M phases of the cell cycle were increased by the treatment with GMCSF. Moreover, anti-GMCSF antibody added to MDMD/MDMDF from the initiation of the primary culture (in the presence of keratinocytes) inhibited the proliferation of melanoblasts/melanocytes as well as the differentiation of melanocytes. Enzyme-linked immunosorbent assay of culture media revealed that GMCSF was secreted from keratinocytes, but not from melanocytes. These results suggest that GMCSF is one of the keratinocyte-derived factors involved in regulating the proliferation and differentiation of neonatal mouse epidermal melanoblasts/melanocytes in culture in cooperation with cAMP elevator and bFGF.     

Plakophilin-2 and the migration,differentiation and transformation of cells derived from the epicardium of neonatal rat hearts

Abstract

During development, epicardial cells act as progenitors for a large fraction of non-myocyte cardiac cells. Expression and function of molecules of the desmosome in the postnatal epicardium has not been studied. The objective of this study was to assess the expression of desmosomal molecules, and the functional importance of the desmosomal protein plakophilin-2 (PKP2), in epicardial and epicardium-derived cells. Epicardial explants were obtained from neonatal rat hearts. Presence of mechanical junction proteins was assessed by immunocytochemistry. Explants after PKP2 knockdown showed increased abundance of alpha smooth muscle actin-positive cells, increased abundance of lipid markers, enhanced cell migration velocity and increased abundance of a marker of cell proliferation. We conclude that a population of non-excitable, cardiac-resident cells express desmosomal molecules and, in vitro, show functional properties (including lipid accumulation) that depend on PKP2 expression. The possible relevance of our data to the pathophysiology of arrhythmogenic right ventricular cardiomyopathy, is discussed.     

Enhanced antimicrobial activity of novel synthetic peptides derived from vejovine and hadrurin

Background

Microbial antibiotic resistance is a challenging medical problem nowadays. Two scorpion peptides displaying antibiotic activity: hadrurin and vejovine were taken as models for the design of novel shorter peptides with similar activity.

Methods

Using the standard Fmoc-based solid phase synthesis technique of Merrifield twelve peptides (18 to 29 amino acids long) were synthesized, purified and assayed against a variety of multi-drug resistant Gram-negative bacteria from clinical isolates. Hemolytic and antiparasitic activities of the peptides and their possible interactions with eukaryotic cells were verified. Release of the fluorophore calcein from liposomes treated with these peptides was measured.

Results

A peptide with sequence GILKTIKSIASKVANTVQKLKRKAKNAVA), and three analogs: Δ(Α29), Δ(K12-Q18; Ν26−Α29), and K4N Δ(K12-Q18; Ν26−Α29) were shown to inhibit the growth of Gram-negative (E. coli ATCC25922) and Gram-positive bacteria (S. aureus), as well as multi-drug resistant (MDR) clinical isolated. The antibacterial and antiparasitic activities were found with peptides at 0.78 to 25 μM and 5 to 25 μM concentration, respectively. These peptides have low cytotoxic and hemolytic activities at concentrations significantly exceeding their minimum inhibitory concentrations (MICs), showing values between 40 and 900 μM for their EC₅₀, compared to the parent peptides vejovine and hadrurin that at the same concentration of their MICs lysed more than 50% of human erythrocytes cells.

Conclusions

These peptides promise to be good candidates to combat infections caused by Gram-negative bacteria from nosocomial infections.

General significance

Our results confirm that well designed synthetic peptides can be an alternative for solving the lack of effective antibiotics to control bacterial infections.     

BRS-3 activation transforms the effect of human bronchial epithelial cells from PGE2 mediated inhibition to TGF-beta1 dependent promotion on proliferation and collagen synthesis of lung fibroblasts

Airway re-modelling in asthma usually results in an irreversible weakness of pulmonary ventilation, however, its initiating or controlling mechanism remains unclear. In this study, we hypothesize that signal communication between airway epithelial cells and sub-mucosal fibroblast cells may play an important role in the maintenance of structure homeostasis in a physiologic condition and in initiation of airway remodelling in a stressed condition. To test the hypothesis, a co-cultured system of human bronchial epithelial cells (BEC) and human lung fibroblasts (HLF) were designed to observe the effects of BEC, in the normal state or in a BRS-3 activated state, on the proliferation and collagen synthesis of HLF. The results showed that the proliferation activities of both BEC and HLF inhibited each other under the normal state. BRS-3-activated BEC can transform the reciprocal inhibition into promoting effects. The secretion of TGF-beta1 increased and the synthesis of PGE2 decreased from BRS-3-activated BEC, which were correlated with the proliferation and collagen synthesis of HLF. The proliferation activities of HLF were weakened by co-culture with TGF-beta1 antisense oligonucleotides (ASO) treated BEC. It was concluded that, in the normal state, BEC inhibits the activities of fibroblasts through release of PGE2 to maintain the airway homeostasis; however when stressed, for example by BRS-3 activation, BEC promote the activities of fibroblasts mediated by TGF-beta1, thereby facilitating the airway re-modelling.     

PPAR‐β facilitating maturation of hepatic‐like tissue derived from mouse embryonic stem cells accompanied by mitochondriogenesis and membrane potential retention

Relatively little is known about mitochondria metabolism in differentiating embryonic stem (ES) cells. Present research focused on several elements of cellular energy metabolism in hepatic‐like tissue derived from mouse ES cells. We demonstrated that mitochondrial location patterns and mitochondrial membrane potential (ΔΨ_m) existed in subsequent differentiation of the tissue. Mitochondriogenesis appeared at the early stage and kept a normal ΔΨ_m in differentiated mature hepatocytes. Peroxisome proliferator‐activated receptor‐α (PPAR‐α) expression was transitorily increased at the beginning, and kept a relatively low level later, which accompanied by expression of PPAR‐γ coactivator (PGC)‐1α, a master regulator of mitochondrial biogenesis. PPAR‐β expression showed robust up‐regulation in the late differentiation course. Enhanced co‐expressions of PPAR‐β and albumin with catalysis of UDP‐glucuronosyltransferases (UGTs) were observed at mature stage. While PPAR‐γ expression changed little before and after differentiation. Mitochondriogenesis could be accelerated by PPAR‐α specific agonist WY14643 and abolished by its antagonist GW6471 at the early stage. Neither of them affected mitochondrial ΔΨ_m and albumin generation in the differentiated hepatocytes. Furthermore, maturation of hepatic‐like tissue and mitochondriogenesis in hepatocyte could be efficiently stimulated by PPAR‐β specific agonist L165041 and abolished by PPAR‐β specific antagonist GSK0660, but not affected by PPAR‐γ specific agonist GW1929. In conclusion, the derived hepatic tissue morphologically possessed cellular energy metabolism features. PPAR‐α seemed only necessary for early mitochondriogenesis, while less important for ΔΨ_m retention in the mature tissue derived. The stimulation of PPAR‐β but not ‐γ enhanced hepatogenesis, hepatocytes maturation, and mitochondriogenesis. PPAR‐β took an important role in cellular energy metabolism of hepatogenesis. J. Cell. Biochem. 109: 498–508, 2010. © 2009 Wiley‐Liss, Inc.     

Crystal structures of mouse 17alpha-hydroxysteroid dehydrogenase (apoenzyme and enzyme-NADP(H) binary complex): identification of molecular determinants responsible for the unique 17alpha-reductive activity of this enzyme

Very recently, the mouse 17alpha-hydroxysteroid dehydrogenase (m17alpha-HSD), a member of the aldo-keto reductase (AKR) superfamily, has been characterized and identified as the unique enzyme able to catalyze efficiently and in a stereospecific manner the conversion of androstenedione (Delta4) into epitestosterone (epi-T), the 17alpha-epimer of testosterone. Indeed, the other AKR enzymes that significantly reduce keto groups situated at position C17 of the steroid nucleus, the human type 3 3alpha-HSD (h3alpha-HSD3), the human and mouse type 5 17beta-HSD, and the rabbit 20alpha-HSD, produce only 17beta-hydroxy derivatives, although they possess more than 70% amino acid identity with m17alpha-HSD. Structural comparisons of these highly homologous enzymes thus offer an excellent opportunity of identifying the molecular determinants responsible for their 17alpha/17beta-stereospecificity. Here, we report the crystal structure of the m17alpha-HSD enzyme in its apo-form (1.9 A resolution) as well as those of two different forms of this enzyme in binary complex with NADP(H) (2.9 A and 1.35 A resolution). Interestingly, one of these binary complex structures could represent a conformational intermediate between the apoenzyme and the active binary complex. These structures provide a complete picture of the NADP(H)-enzyme interactions involving the flexible loop B, which can adopt two different conformations upon cofactor binding. Structural comparison with binary complexes of other AKR1C enzymes has also revealed particularities of the interaction between m17alpha-HSD and NADP(H), which explain why it has been possible to crystallize this enzyme in its apo form. Close inspection of the m17alpha-HSD steroid-binding cavity formed upon cofactor binding leads us to hypothesize that the residue at position 24 is of paramount importance for the stereospecificity of the reduction reaction. Mutagenic studies have showed that the m17alpha-HSD(A24Y) mutant exhibited a completely reversed stereospecificity, producing testosterone only from Delta4, whereas the h3alpha-HSD3(Y24A) mutant acquires the capacity to metabolize Delta4 into epi-T.     

Queuosine deficient tRNAHis and tRNAAsp from the spleens of young mice,erythroleukemic tumoral spleens and cultured Friend cells

tRNAs were isolated from the spleens of young mice, erythroleukemic spleens and cultured Friend cells. Queuosine (Q) deficient tRNAs were labelled in their anticodon with radioactive guanine using the exchange reaction catalyzed by E. coli tRNA-guanine transglycosylase. tRNAs were then specifically aminoacylated. In both normal and tumoral spleen (TF-P10), tRNA^His was the main guanine containing (G) isoacceptor as shown by RPC-5 chromatography. In vitro, the tumor derived cell line (TF-P10c) retained the G-tRNA^His species while Friend cell line, clone 707 (FLC), did not. A common feature found in both cultured cell lines was the high level of G-tRNA^Asp. The meaning of the relative abundance of Q-deficient tRNA^His and of tRNA^Asp observed in transformed cells of erythroid origin is discussed.     

Structural and biochemical analysis of the Rv0805 cyclic nucleotide phosphodiesterase from Mycobacterium tuberculosis

Cyclic nucleotide monophosphate (cNMP) hydrolysis in bacteria and eukaryotes is brought about by distinct cNMP phosphodiesterases (PDEs). Since these enzymes differ in amino acid sequence and properties, they have evolved by convergent evolution. Cyclic NMP PDEs cleave cNMPs to NMPs, and the Rv0805 gene product is, to date, the only identifiable cNMP PDE in the genome of Mycobacterium tuberculosis. We have shown that Rv0805 is a cAMP/cGMP dual specificity PDE, and is unrelated in amino acid sequence to the mammalian cNMP PDEs. Rv0805 is a dimeric, Fe(3+)-Mn(2+) binuclear PDE, and mutational analysis demonstrated that the active site metals are co-ordinated by conserved aspartate, histidine and asparagine residues. We report here the structure of the catalytic core of Rv0805, which is distantly related to the calcineurin-like phosphatases. The crystal structure of the Rv0805 dimer shows that the active site metals contribute to dimerization and thus play an additional structural role apart from their involvement in catalysis. We also present the crystal structures of the Asn97Ala mutant protein that lacks one of the Mn(2+) co-ordinating residues as well as the Asp66Ala mutant that has a compromised cAMP hydrolytic activity, providing a structural basis for the catalytic properties of these mutant proteins. A molecule of phosphate is bound in a bidentate manner at the active site of the Rv0805 wild-type protein, and cacodylate occupies a similar position in the crystal structure of the Asp66Ala mutant protein. A unique substrate binding pocket in Rv0805 was identified by computational docking studies, and the role of the His140 residue in interacting with cAMP was validated through mutational analysis. This report on the first structure of a bacterial cNMP PDE thus significantly extends our molecular understanding of cAMP hydrolysis in class III PDEs.     

Histidine-rich protein Hpn from Helicobacter pylori forms amyloid-like fibrils in vitro and inhibits the proliferation of gastric epithelial AGS cells

Helicobacter pylori causes various gastric diseases, such as gastritis, peptic ulcerations, gastric cancer and mucosa-associated lymphoid tissue lymphoma. Hpn is a histidine-rich protein abundant in this bacterium and forms oligomers in physiologically relevant conditions. In this present study, Hpn oligomers were found to develop amyloid-like fibrils as confirmed by negative stain transition electron microscopy, thioflavin T and Congo red binding assays. The amyloid-like fibrils of Hpn inhibit the proliferation of gastric epithelial AGS cells through cell cycle arrest in the G2/M phase, which may be closely related to the disruption of mitochondrial bioenergetics as reflected by the significant depletion of intracellular ATP levels and the mitochondrial membrane potential. The collective data presented here shed some light on the pathologic mechanisms of H. pylori infections.