Differential regulation of FBXW7 isoforms by various stress stimuli

Fbxw7 is a tumor suppressor mutated in a wide range of human cancers. It serves as the substrate recognition component of SCF E3 ubiquitin ligases, and intensive effort was made to identify its substrates. Some of the substrates are central regulators of the cell cycle, cell fate determination, and cellular survival. Unlike the many efforts aimed at identifying novel targets, little is known about the regulation of Fbw7 isoform expression. In this study, we examined the mRNA expression of different FBXW7 isoforms during the cell cycle and after exposure to various stress stimuli. We observed that Fbw7β is induced by all the stress stimuli tested, mostly, but not exclusively, in a p53-dependent manner. In fact, FBXW7β was found to be the most potently induced p53 target gene in HCT-116 cells. Expression of FBXWα and γ is p53-independent and their responsiveness to most stress stimuli is limited. Furthermore, their pattern of stress responsiveness is very different from that of the β isoform. Under certain conditions, the same genotoxic agent stimulates induction of β and repression of α. Analysis of FACS-sorted cells in specific phases of the cell cycle by using fluorescent ubiquitination-based cell cycle indicator (FUCCI), showed a significant repression of the γ isoform during the S phase of normal cycling HCT-116 cells. Altogether, this study suggests differential regulation of the 3 Fbw7 isoforms.     

Capacity of human beta-defensin expression in gene-transduced and cytokine-induced cells

The purpose of this study was to determine the capacity of cells transduced with human beta-defensins (HBDs) to express antimicrobial peptides, since sufficient expression level is required for effective antimicrobial activity. Retroviral vector pBabeNeo and lentiviral vector SIN18cPPTRhMLV (SIN18) carrying HBDs were utilized to transduce non-HBD-expressing cells such as fibroblasts or HBD-producing oral epithelial cells. We found that HBD-3 gene transfer to fibroblasts was possible not via retrovirus but by direct vector transfection. SIN18 had high transduction efficiencies (80.9-99.9%) and transduced cells expressed higher amounts of HBD-2 than those by pBabeNeo. Primary human gingival epithelial cells (HGECs) expressed greater amounts of HBD-2 than primary fibroblasts after lentiviral transduction. Additionally, HBD-2 secretion from transduced HGECs cells was further increased when stimulated with IL-1 or TNFalpha. Our data indicate that while HBD-2 expression is limited in primary fibroblasts, its expression in HGECs may be maximized by gene transduction plus cytokine induction.     

Differential regulation of Na+/H+ antiporter gene expression in vascular smooth muscle cells by hypertrophic and hyperplastic stimuli

The Na+/H+ antiporter is a ubiquitous transmembrane protein that plays a vital role in cell growth via regulation of intracellular Na+ and H+. In vascular smooth muscle cells (VSMC), vasoconstrictors and mitogens rapidly activate the antiporter, suggesting that both should have growth promoting effects. Indeed, angiotensin II increases VSMC protein and volume (hypertrophy), but does not increase cell number (hyperplasia). In the present work we investigated whether alterations in the steady state levels of Na+/H+ antiporter mRNA might differentiate these VSMC growth responses. Differences in function of the Na+/H+ antiporter appeared likely because exposure of growth-arrested VSMC for 24 h to 100 nM angiotensin II decreased intracellular pH from 7.08 to 6.99, while exposure to 10% calf serum caused an increase to 7.18. Simultaneous measurement of Na+/H+ antiporter mRNA levels, using the human c28 cDNA, revealed a 25-fold increase in response to serum (as well as to platelet-derived and fibroblast growth factors), but no change in response to angiotensin II. All agonists increased mRNA levels of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase approximately 3-fold. The increase in Na+/H+ antiporter mRNA induced by serum was first apparent within 2 h and peaked 24 h after treatment. These results demonstrate that expression of Na+/H+ antiporter mRNA in VSMC is dependent on growth state: hyperplastic agonists (serum, platelet-derived and fibroblast growth factor) increase the steady state levels of Na+/H+ antiporter mRNA while a hypertrophic agonist (angiotensin II) does not.     

Production of bioactive human beta-defensin 5 and 6 in Escherichia coli by soluble fusion expression

This work reports the first successful recombinant expression and purification of human beta-defensin 5 (HBD5) and human beta-defensin 6 (HBD6) in Escherichia coli. HBD5 and HBD6 are cationic antimicrobial peptides with three conserved cysteine disulfide bonds. Two codon-optimized sequences coding the HBD5 gene (sHBD5) and HBD6 gene (sHBD6), respectively, were synthesized, and each gene fused with thioredoxin A (TrxA) to construct the expression vectors. The plasmids were transformed into E. coli BL21 (DE3) strains and cultured in MBL medium, which gave high volumetric productivity of HBD5 and HBD6 fusion proteins of up to 1.49 g L⁻¹ and 1.57 g L⁻¹, respectively. Soluble HBD5 and HBD6 fusion proteins account for 95.2% and 97.6% of the total fusion proteins, respectively. After cell disruption, the soluble fusion proteins were recovered by affinity chromatography and cleaved by enterokinase. Pure HBD5 and HBD6 were recovered using cationic exchange chromatography. The overall recoveries of HBD5 and HBD6 were 38% and 35%, respectively. Importantly, both HBD5 and HBD6 products showed antimicrobial activity against E. coli but not Staphylococcus aureus. Antimicrobial activity against E. coli of both HBD5 and HBD6 were suppressed by NaCl.     

人体皮肤微生物群落研究进展

皮肤作为人体最大的器官,上面定居着各种各样的微生物,它们大部分是无害的,甚至对人体有益。皮肤表面的生态环境因不同的表面特征和外部因素而呈现不同的格局,使得分布于皮肤上的微生物群落出现差异。分子生物学技术的发展使研究皮肤表面微生物群落的高度多样性和多变性成为可能,而且可从生态系统角度去理解和认识皮肤微生物。本文就皮肤微生物群落的主要特点、微生物群落与疾病的联系及其具体应用等方面作一综述。     

Differential regulation of cadherins by dexamethasone in human osteoblastic cells

Human osteoblasts express a repertoire of cadherins, including N-cadherin (N-cad), cadherin-11 (C11), and cadherin-4 (C4). We have previously shown that direct cell-cell adhesion via cadherins is critical for BMP-2-induced osteoblast differentiation. In this study, we have analyzed the regulation of cadherin expression in normal human trabecular bone osteoblasts (HOB), and osteoprogenitor marrow stromal cells (BMC), during exposure to dexamethasone, another inducer of human bone cell differentiation. Dexamethasone inhibited the expression of both C11 and N-cad mRNA in both BMC and HOB, although the effect was much more pronounced on N-cad than on C11. This action of the steroid was dose dependent, was maximal at 10(-7) M concentration, and occurred as early as after 1 day of incubation. By contrast, expression of C4 mRNA and protein was strongly induced by dexamethasone in BMC and was stimulated in HOB. This stimulatory effect lasted for at least 2 weeks of incubation. A cadherin inhibitor, HAV-containing decapeptide only partially ( approximately 50%) prevented dexamethasone-induced stimulation of alkaline phosphatase activity by BMC, which instead was not altered by incubation with a neutralizing antibody against C4. Therefore, the pattern of cadherin regulation by dexamethasone radically differs form that observed with BMP-2. Dexamethasone effects on certain osteoblast differentiated features, such as induction of alkaline phosphatase activity are not strictly dependent on cadherin function.     

Lipopolysaccharide-induced IL-1 beta production by human uterine macrophages up-regulates uterine epithelial cell expression of human beta-defensin 2

The uterine endometrium coordinates a wide spectrum of physiologic and immunologic functions, including endometrial receptivity and implantation as well as defense against sexually transmitted pathogens. Macrophages and epithelial cells cooperatively mediate innate host defense against bacterial invasion through the generation of immunologic effectors, including cytokines and antimicrobial peptides. In this study, we demonstrate that stimulation of peripheral blood monocytes and uterine macrophages with bacterial LPS induces the production of biologically active proinflammatory IL-1beta. High doses of estradiol enhance LPS-induced IL-1beta expression in an estrogen receptor-dependent manner. Furthermore, both peripheral blood monocyte- and uterine macrophage-derived IL-1beta induce secretion of antimicrobial human beta-defensin 2 by uterine epithelial cells. These data indicate dynamic immunologic interaction between uterine macrophages and epithelial cells and implicate a role for estradiol in the modulation of the immune response.     

Soluble fusion expression and characterization of bioactive human beta-defensin 26 and 27

This study reports the first successful recombinant expression of cationic antimicrobial peptides human beta-defensin-26 and human beta-defensin-27 in Escherichia coli. HBD26 and HBD27 genes were synthesized through codon optimization, and each gene was then cloned into the expression vector pET32, which feature fusion protein thioredoxin at the N-terminal. The recombinant plasmids were then transformed into E. coli BL21 (DE3) and cultured in MBL medium, which gave yields of HBD26 and HBD27 fusion proteins of up to 1.38 and 1.29 g l⁻¹, respectively. Affinity chromatography was used to purify the soluble fusion proteins, and the N-terminal TrxA tags were cleaved off by enterokinase. Pure HBD26 and HBD27 were then obtained by cationic exchange chromatography. The overall recovery of HBD26 was 38% and that of HBD27 reached 36%. Both variants showed salt-sensitive antimicrobial activity against gram-negative E. coli but not against gram-positive Staphylococcus aureus and Saccharomyces cerevisiae.     

Differential expression of β-glucosidase in tomato — stress stimuli systems

In the present work the responses of β-glucosidase in leaves of tomato plants subjected to various stress factors of both pathogenic (fungi, bacteria, viruses) and abiotic origin (heat shock) were studied. Biochemical and cytochemical methods were applied. It was established that an increase of β-glucosidase activity is induced uniquely by fungal pathogens. The cytochemical tests confirm the finding. Hence, the conclusion can be drawn that β-glucosidase response is a specific character of fungal pathogenesis in tomato; probably, the enzyme is involved in plant — fungi recognition. The data are in accordance with our previous results on tobacco and wheat — stress stimuli systems.     

Differential regulation of neuregulin 1 expression by progesterone in astrocytes and neurons

Glial-neuronal interactions are crucial processes in neuromodulation and synaptic plasticity. The neuregulin 1 family of growth and differentiation factors have been implicated as bidirectional signaling molecules that are involved in mediating some of these interactions. We have shown previously that neuregulin 1 expression is regulated by the gonadal hormones progesterone and 17beta-estradiol in the CNS, which might represent a novel, indirect mechanism of the neuromodulatory actions of these gonadal hormones. In the present study, we sought to determine the effects of progesterone and 17beta-estradiol on neuregulin 1 expression in rat cortical astrocytes and neurons in vitro. We observed that progesterone increased the expression of neuregulin 1 mRNA and protein in a dose-dependent manner in cultured astrocytes, which was blocked by the progesterone receptor antagonist RU-486. In contrast, 17beta-estradiol did not increase either neuregulin 1 mRNA or protein in astrocytes. We observed no effect of either progesterone or 17beta-estradiol on neuregulin 1 mRNA and protein in rat cortical neurons in vitro. Finally, we observed that treatment of cortical neurons with recombinant NRG1-beta1 caused PSD-95 to localize in puncta similar to that observed following treatment with astrocyte-conditioned medium. These results demonstrate that progesterone regulates neuregulin 1 expression, principally in astrocytes. This might represent a novel mechanism of progesterone-mediated modulation of neurotransmission through the regulation of astrocyte-derived neuregulin 1.