Enhanced epithelial-mesenchymal transition-like phenotype in N-acetylglucosaminyltransferase V transgenic mouse skin promotes wound healing

N-Acetylglucosaminyltransferase V (GnT-V) catalyzes the β1,6 branching of N-acetylglucosamine on N-glycans. GnT-V expression is elevated during malignant transformation in various types of cancer. However, the mechanism by which GnT-V promotes cancer progression is unclear. To characterize the biological significance of GnT-V, we established GnT-V transgenic (Tg) mice, in which GnT-V is regulated by a β-actin promoter. No spontaneous cancer was detected in any organs of the GnT-V Tg mice. However, GnT-V expression was up-regulated in GnT-V Tg mouse skin, and cultured keratinocytes derived from these mice showed enhanced migration, which was associated with changes in E-cadherin localization and epithelial-mesenchymal transition (EMT). Further, EMT-associated factors snail, twist, and N-cadherin were up-regulated, and cutaneous wound healing was accelerated in vivo. We further investigated the detailed mechanisms of EMT by assessing EGF signaling and found up-regulated EGF receptor signaling in GnT-V Tg mouse keratinocytes. These findings indicate that GnT-V overexpression promotes EMT and keratinocyte migration in part through enhanced EGF receptor signaling.     

Polycystin-1 may play an important role in mechanical modulation of bone growth and healing

Mechanical stress is known to modulate bone growth and healing. However, the mechanisms underlying the mechanotransduction are not fully understood. Previous studies show that PC1 is a promising candidate among proteins that may play a role in the mechanotransduction process as it has been shown to function as a flow sensor in renal epithelium and it is known to be important for the growth of for skeletal development. We hypothesized that PC1 plays an important role in bone responses to mechanical stress. PC1 is required for the proliferation, differentiation and survival of periosteal osteochondroprogenitor cells upon mechanical stimulation of bone. Using both genetically manipulated animal models and animals undergoing are necessary to test this hypothesis.     

毛脚鵟细胞体外培养体系建立及三种组织来源细胞特性分析

用组织块培养法对毛脚鵟不同组织进行原代培养,获得了3种不同组织来源的细胞,并成功对细胞进行了冷冻保存和复苏。在传代培养过程中,对比分析了3种组织来源细胞的形态学、生长曲线、贴壁率、核型等生物学特性。形态学方面,3种来源细胞均为成纤维样细胞。对于3种组织来源细胞的贴壁能力分析显示,输卵管源细胞最强,肺源细胞和气管源细胞次之。3种不同组织来源细胞的倍增时间分别为(29.91±0.39)、(33.18±0.21)和(30.67±0.28)h,群体倍增次数分别为3.54±0.01、4.52±0.02和4.38±0.03。毛脚鵟细胞的染色体数目为2n=68,性染色体为典型的ZW型。本实验为今后毛脚鵟细胞利用、遗传信息的保存及生物学特性的深入研究提供实验材料和依据。     

Epstein-Barr virus-transformed B lymphocytes produce low molecular mass molecules with autocrine growth factor and competence factor activity

A human Epstein-Barr virus (EBV)-positive lymphoblastoid B cell line, named BA-D10-4, produces a factor of a molecular mass less than 10 kDa that promotes cell proliferation of both BA-D10-4 cells and other human T or B lymphoid cell lines, either EBV-positive or -negative. The factor synergizes with higher molecular mass autocrine growth factors and makes both BA-D10-4 cells and B cell lines from Burkitt's lymphoma, but not cells from T cell leukemia, more responsive to interleukin-1 and interleukin-6. Therefore, this low molecular mass factor seems to be an autocrine growth factor per se and to have the characteristics of a competence factor.     

Comparative modeling and analysis of microfluidic and conventional DNA microarrays

A theoretical analysis was developed to predict molecular hybridization rates for microarrays where samples flow through microfluidic channels and for conventional microarrays where samples remain stationary during hybridization. The theory was validated by using a multiplexed microfluidic microarray where eight samples were hybridized simultaneously against eight probes using 60-mer DNA strands. Mass transfer coefficients ranged over three orders of magnitude where either kinetic reaction rates or molecular diffusion rates controlled overall hybridization rates. Probes were printed using microfluidic channels and also conventional spotting techniques. Consistent with the theoretical model, the microfluidic microarray demonstrated the ability to print DNA probes in less than 1 min and to detect 10-pM target concentrations with hybridization times in less than 5 min.     

Human blood-derived fibrin releasates: Composition and use for the culture of cell lines and human primary cells

We have evaluated the capacity of two human blood fractions to substitute for FBS as growth medium supplement for human and animal cell cultures. Non-anticoagulated blood from volunteer donors (N = 13) was centrifuged to isolate a supernatant serum (SS) and a platelet-rich fibrin (PRF) clot which was squeezed to extract the releasate (PRFR). Both materials were characterized for the content in PDGF-AB, TGF-β1, VEGF, bFGF, EGF, IGF, total protein, albumin, IgG, IgM IgA, fibrinogen, cholesterol, triglycerides, various chemistry analytes and hemoglobin. Cell growth promoting activity of pooled SS and PRFR at 1, 5, and 10% in growth medium was evaluated over 7 days using human (HEK293, MG-63) and animal (SIRC, 3T3) cell lines and two human primary cultures (gingival fibroblasts and periodontal ligaments). Viable cell count was compared to that in cultures in FBS free-medium and 10% FBS supplement. SS and PRFR at 1-10% stimulated cell growth significantly more than FBS-free medium and in a way similar to 10% FBS in all cultures apart from 3T3. These two human blood-derived fibrin releasates are equally efficient to substitute for FBS as supplement for cell cultures and could be useful for specialized applications in regenerative medicine, dentistry and oral implantology, or cell therapy.     

Control of megakaryocyte expansion and bone marrow fibrosis by lysyl oxidase

Lysyl oxidase (LOX), a matrix cross-linking protein, is known to be selectively expressed and to enhance a fibrotic phenotype. A recent study of ours showed that LOX oxidizes the PDGF receptor-β (PDGFR-β), leading to amplified downstream signaling. Here, we examined the expression and functions of LOX in megakaryocytes (MKs), the platelet precursors. Cells committed to the MK lineage undergo mitotic proliferation to yield diploid cells, followed by endomitosis and acquisition of polyploidy. Intriguingly, LOX expression is detected in diploid-tetraploid MKs, but scarce in polyploid MKs. PDGFR-BB is an inducer of mitotic proliferation in MKs. LOX inhibition with β-aminopropionitrile reduces PDGFR-BB binding to cells and downstream signaling, as well as its proliferative effect on the MK lineage. Inhibition of LOX activity has no influence on MK polyploidy. We next rationalized that, in a system with an abundance of low ploidy MKs, LOX could be highly expressed and with functional significance. Thus, we resorted to GATA-1(low) mice, where there is an increase in low ploidy MKs, augmented levels of PDGF-BB, and an extensive matrix of fibers. MKs from these mice display high expression of LOX, compared with control mice. Importantly, treatment of GATA-1(low) mice with β-aminopropionitrile significantly improves the bone marrow fibrotic phenotype, and MK number in the spleen. Thus, our in vitro and in vivo data support a novel role for LOX in regulating MK expansion by PDGF-BB and suggest LOX as a new potential therapeutic target for myelofibrosis.     

HIV and bone disease

Advances in management have resulted in a dramatic decline in mortality for individuals infected with human immunodeficiency virus (HIV). This decrease in mortality, initially the result of improved prophylaxis and treatment of opportunistic infections but later mediated by the use of highly-active antiretroviral therapy (HAART) has led to the need to consider long-term complications of the disease itself, or its treatment. Bone disease is increasingly recognised as a concern.The prevalence of reduced BMD and possibly also fracture incidence are increased in HIV-positive individuals compared with HIV-negative controls. There are many potential explanations for this - an increased prevalence of established osteoporosis risk factors in the HIV-positive population, a likely direct effect of HIV infection itself and a possible contributory role of ARV therapy. At present, the assessment of bone disease and fracture risk remains patchy, with little or no guidance on identifying those at increased risk of reduced BMD or fragility fracture. Preventative and therapeutic strategies with bone specific treatments need to be developed. Limited data suggest bisphosphonates may be beneficial in conjunction with vitamin D and calcium supplementation in the treatment of reduced BMD in HIV-infected patients but larger studies of longer duration are needed. The safety and cost-effectiveness of these and other treatments needs to be evaluated.     

Stem cells and growth factor delivery systems for cardiovascular disease

Coronary (CAD) and peripheral (PAD) artery diseases are major causes of morbidity and mortality, and millions of CAD and PAD patients are treated by various medications, bypass surgery or angioplasty around the world. Such patients might benefit from novel stem cells and tissue engineering strategies aimed at accelerating natural processes of postnatal collateral vessel formation and repairing damaged tissues. By combining three fundamental “tools”, namely stem cells, biomaterials and growth factors (GFs), such strategies may enhance the efficacy of cell therapy in several ways: (a) by supplying exogenous stem cells or GFs that stimulate resident cardiac stem cell (CSC) migration, engraftment and commitment to cardiomyocytes, and that induce and modulate arterial response to ischemia; (b) by supporting the maintenance of GFs and transplanted stem cells in the damaged tissues through the use of biocompatible and biodegradable polymers for a period of time sufficient to allow histological and anatomical restoration of the damaged tissue. This review will discuss the potential of combining stem cells and new delivery systems for growth factors, such as vehicle-based delivery strategies or cell-based gene therapy, to facilitate regeneration of ischemic tissues. These approaches would promote the ability of resident CSCs or of exogenous multipotent stem cells such as adipose tissue-derived mesenchymal stem cells (AT-MSCs) to induce the healing of damaged tissue, by recruiting and directing these cells into the damage area and by improving angiogenesis and reperfusion of ischemic tissues.     

m-calpain Activation Is Regulated by Its Membrane Localization and by Its Binding to Phosphatidylinositol 4,5-Bisphosphate

m-calpain plays a critical role in cell migration enabling rear de-adhesion of adherent cells by cleaving structural components of the adhesion plaques. Growth factors and chemokines regulate keratinocyte, fibroblast, and endothelial cell migration by modulating m-calpain activity. Growth factor receptors activate m-calpain secondary to phosphorylation on serine 50 by ERK. Concurrently, activated m-calpain is localized to its inner membrane milieu by binding to phosphatidylinositol 4,5-bisphosphate (PIP₂). Opposing this, CXCR3 ligands inhibit cell migration by blocking m-calpain activity secondary to a PKA-mediated phosphorylation in the C2-like domain. The failure of m-calpain activation in the absence of PIP₂ points to a key regulatory role, although whether this PIP₂-mediated membrane localization is regulatory for m-calpain activity or merely serves as a docking site for ERK phosphorylation is uncertain. Herein, we report the effects of two CXCR3 ligands, CXCL11/IP-9/I-TAC and CXCL10/IP-10, on the EGF- and VEGF-induced redistribution of m-calpain in human fibroblasts and endothelial cells. The two chemokines block the tail retraction and, thus, the migration within minutes, preventing and reverting growth factor-induced relocalization of m-calpain to the plasma membrane of the cells. PKA phosphorylation of m-calpain blocks the binding of the protease to PIP₂. Unexpectedly, we found that this was due to membrane anchorage itself and not merely serine 50 phosphorylation, as the farnesylation-induced anchorage of m-calpain triggers a strong activation of this protease, leading notably to an increased cell death. Moreover, the ERK and PKA phosphorylations have no effect on this membrane-anchored m-calpain. However, the presence of PIP₂ is still required for the activation of the anchored m-calpain. In conclusion, we describe a novel mechanism of m-calpain activation by interaction with the plasma membrane and PIP₂ specifically, this phosphoinositide acting as a cofactor for the enzyme. The phosphorylation of m-calpain by ERK and PKA by growth factors and chemokines, respectively, act in cells to regulate the enzyme only indirectly by controlling its redistribution.     

Wound healing enhancement in leg ulcers: A case report

Wound healing in the skin is a complex biological process in which numerous types of cells, cytokines, growth factors, proteases and extracellular matrix components act in concert to restore the integrity of injured tissue. Cultivated allogenic human keratinocytes have been used for the treatment of various skin defects like burnwounds, surgical wounds, in exfoliative skin diseases and chronic wounds. A new method for wound healing enhancement in leg ulcers using cultured allogenic keratinocytes suspended in fibrin glue and used in spray technique is introduced. Allogenic keratinocytes are supposed to enhance granulation tissue production and to stimulate reepithelisation due to their release of growth factors and thus are able to recreate an active wound. This revised version was published online in July 2006 with corrections to the Cover Date.     

Characterization and Partial Purification of AIM: A Plasma Protein That Induces Rat Cerebral Type 2 Astroglia from Bipotential Glial Progenitors

Studies on glial cultures have demonstrated that fetal bovine serum contains a factor that induces bipotential glial precursors known as oligodendrocyte-type 2 astrocyte (O-2A) progenitors to become type 2 astroglia rather than oligodendroglia. The goal of this research project was to characterize and purify this factor, which we refer to as the astroglia-inducing molecule (AIM). Using cultures enriched in O-2A progenitors, we determined that AIM is present in human and bovine sera and that fetal bovine serum qualified as the best serum for purifying AIM. AIM is heat and trypsin labile and may be a plasma glycoprotein. A 240-fold enriched AIM preparation was produced by applying an ammonium sulfate precipitate of fetal bovine serum to heparin and then lentil lectin-agarose, followed by gel filtration chromatography. In crude preparations, AIM activity migrated at 50 kDa by gel filtration. With enrichment, activity was seen at several molecular masses, all of which were approximate multiples of 50 kDa. Treatment with 6 M guanidine hydrochloride generated an AIM with a molecular mass between 12 and 18 kDa, a result suggesting that AIM aggregates. On a preparative isoelectric focusing gel, AIM activity most frequently migrated between pH values of 3 and 4; however, proteins with isoelectric points of greater than 9 or at 6 also had activity in several experiments. These data suggest that either multiple AIMs exist or that a single AIM exists that associates with other proteins. Immunofluorescence for ganglioside GD3 and glial fibrillary acidic protein confirmed that AIM preparations induce type 2 astroglia from O-2A progenitors and suggests that AIM has little effect on type 1 astroglia. Because none of the known growth factors that have been tested to date mimics its effects. AIM may be a novel differentiation factor.     

Non-neuronal cells of rat hypothalamus in dissociated cell culture

Summary The neurophysin that is biosynthesised in association with the neurohypophysial hormone vasopressin (vasopressin-neurophysin) affects the growth and DNA synthesis of rat hypothalamic non-neuronal cells in culture. Over a narrow range of concentrations vasopressin-neurophysin stimulated growth, as assessed by increase in cell numbers, about five-fold, in conditions where fetal calf serum concentration was limiting (0.2% fetal calf serum). Maximum stimulation occurred in the presence of 20 to 30 ng vasopressin-neurophysin per ml of medium. DNA synthesis was increased by a factor of three in the presence of 30 ng vasopressin-neurophysin per ml of medium. At least two populations of non-neuronal hypothalamic cells were present in the cultures, and these were both affected by vasopressin-neurophysin.This study allows the suggestion that neurophysin may be acting as a growth-regulating factor at its release site, playing a part in the interactions of neurones and glial cells in the hypothalamo-neurohypophysial system.     

重组骨形态发生蛋白-7在骨损伤疾病治疗中的研究进展

骨形态发生蛋白-7(BMP-7)是具有强诱骨活性的蛋白质因子,已通过基因工程技术在体外得到表达,较长时间以来不断被应用于骨损伤疾病的研究,得到了确切的治疗效果。通过载体将BMP-7基因转入真核细胞,与生物聚合载体复合后植入体内,能表达并分泌活性的BMP-7,诱导骨细胞的生成,促进骨组织的修复,成为一种新的有效的治疗手段。     

The transforming growth factor-betas: structure, signaling, and roles in nervous system development and functions

Transforming growth factor-betas (TGF-betas) are among the most widespread and versatile cytokines. Here, we first provide a brief overview of their molecular biology, biochemistry, and signaling. We then review distribution and functions of the three mammalian TGF-beta isoforms, beta1, beta2, and beta3, and their receptors in the developing and adult nervous system. Roles of TGF-betas in the regulation of radial glia, astroglia, oligodendroglia, and microglia are addressed. Finally, we review the current state of knowledge concerning the roles of TGF-betas in controlling neuronal performances, including the regulation of proliferation of neuronal precursors, survival/death decisions, and neuronal differentiation.     

Roles of cell-intrinsic and microenvironmental factors in photoreceptor cell differentiation

Photoreceptor differentiation requires the coordinated expression of numerous genes. It is unknown whether those genes share common regulatory mechanisms or are independently regulated by distinct mechanisms. To distinguish between these scenarios, we have used in situ hybridization, RT-PCR, and real-time PCR to analyze the expression of visual pigments and other photoreceptor-specific genes during chick embryo retinal development in ovo, as well as in retinal cell cultures treated with molecules that regulate the expression of particular visual pigments. In ovo, onset of gene expression was asynchronous, becoming detectable at the time of photoreceptor generation (ED 5-8) for some photoreceptor genes, but only around the time of outer segment formation (ED 14-16) for others. Treatment of retinal cell cultures with activin, staurosporine, or CNTF selectively induced or down-regulated specific visual pigment genes, but many cognate rod- or cone-specific genes were not affected by the treatments. These results indicate that many photoreceptor genes are independently regulated during development, are consistent with the existence of at least two distinct stages of gene expression during photoreceptor differentiation, suggest that intrinsic, coordinated regulation of a cascade of gene expression triggered by a commitment to the photoreceptor fate is not a general mechanism of photoreceptor differentiation, and imply that using a single photoreceptor-specific "marker" as a proxy to identify photoreceptor cell fate is problematic.