Development of a sensitive bioassay method for quorum sensing inhibitor screening using a recombinantAgrobacterium tumefaciens

Acylhomoserine lactones (AHLs) are known to be the triggering molecules in the quorum sensing mechanism of many gram-negative bacteria. In order to detect AHL inhibitors that are potential biofilm inhibitors, a convenient and sensitive bioassay was developed based on the β-galactosidase activity (β-GAL) of a recombinantAgrobacterium tumefaciens strain. A series of commercially available AHLs were tested for inducing β-GAL at varying concentrations in agar-plate and liquid cultures of the reporter strain. All AHLs tested exhibited a concentration-dependent induction, and octanoyl homoserine lactone (OHL) showed the highest sensitivity with a detection limit of 0.1 nM in the liquid culture assay. When fimbrolide, a known quorum sensing inhibitor, was added, induction of β-GAL by OHL was repressed. The repression at a constant OHL concentration was dependent on the fimbrolide concentration with the detection limit below 1 ppm, indicating that this assay is a sensitive method for screening AHL inhibitors.     

Characterization of gana-1, a Caenorhabditis elegans gene encoding a single ortholog of vertebrate α-galactosidase and α-N-acetylgalactosaminidase

Background  

Human α-galactosidase A (α-GAL) and α-N-acetylgalactosaminidase (α-NAGA) are presumed to share a common ancestor. Deficiencies of these enzymes cause two well-characterized human lysosomal storage disorders (LSD) – Fabry (α-GAL deficiency) and Schindler (α-NAGA deficiency) diseases. Caenorhabditis elegans was previously shown to be a relevant model organism for several late endosomal/lysosomal membrane proteins associated with LSDs. The aim of this study was to identify and characterize C. elegans orthologs to both human lysosomal luminal proteins α-GAL and α-NAGA.     

Gene delivery into neuronal and glial cells by using a replication-deficient adenovirus vector: prospects for neurological gene therapy

We have used a recombinant adenovirus vector (E1−) expressing β-galactosidase to explore a novel mechanism with which to transfer genes into cells of the central nervous system (CNS). The replication-deficient adenovirus vector expressing β-galactosidase (RAd35) was propagated on a permissive helper cell line (293 cells). High level protein expression from the human cytomegalovirus immediate early promoter (hCMV IE) was obtained in a target cell population of RAd35 infected cultured neuronal and glial cell lines. Light microscopy showed that over 50% of the glial cells studied expressed β-galactosidase. Following retinoic acid treatment, RAd35 infected cell lines ND7/23, NG108 and NTera2, showed β-galactosidase expression in up to 90% of the cells. In addition, these cells showed morphological evidence of differentiation into neurons. This pattern of β-galactosidase expression was also observed in primary rat cerebella granule neuron cultures. In vivo studies were performed in Balb/c mice following direct intracranial injections of RAd35 into the brain. Cell sections showed a localised staining in the brain at the site of injection of the virus. Non-replicating adenovirus vectors are therefore highly efficient systems for delivering a transgene into brain cells. However, their broad cell tropism may limit their applications for genetic disorders in which a specific cell type is to be targeted for gene therapy. To address this problem, we have constructed adenovirus vectors which contain specific neuronal promoters and are currently assessing in vitro expression. This revised version was published online in July 2006 with corrections to the Cover Date.     

Laminin α1 domains LG4-5 are essential for the complete differentiation of visceral endoderm

The heterotrimeric basement membrane protein laminin-111 is essential for early mouse embryogenesis. Its β1 and γ1 chains are crucial for endoderm differentiation and for the formation of basement membranes, whereas α1 chain null mice only lack the extraembryonic Reichert’s membrane. Nevertheless, mice deficient in the cell-binding α1 globular domains 4-5 (LG4-5) have a more severe phenotype than animals devoid of the whole α1 chain, as these domains are required for the formation of a polarized ectoderm. However, the influence of the α1LG4-5 domains on endoderm differentiation is unclear. We have used microarray analysis to compare the expression profiles of normal and α1LG4-5-deficient embryoid bodies and show that genes encoding secreted plasma proteins and proteins involved in endocytosis are reduced in α1LG4-5-deficient embryoid bodies, indicating incomplete differentiation of the visceral endoderm. Moreover, mice lacking α1LG4-5 display endoderm disorganization and a defective expression of the endoderm marker Dab2. We hypothesize that α1LG4-5 domains provide an autocrine signal necessary for the complete differentiation of a functional visceral endoderm and vital signals for the polarization of the epiblast.     

Novel mini β-TCP 3D perfusion bioreactor for proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells

Applications of bioreactors in combination with scaffolding materials are promising in tissue-engineering fields. To rapidly produce bone mesenchymal stem cells (MSCs) suitable for osteogenic differentiation (OSD), we developed a novel technique for β-TCP (β-tricalcium phosphate) scaffolds preparation and employed these scaffolds to build a new type of mini three dimensional (3D) perfusion bioreactor. Compared to the 2D static culture, MSCs acquired much higher amplification rate and alkaline phosphatase (ALP) activity over a 24-day culture period. Interestingly, the Specific ALP activity was independent of the growth rate under adequate osteogenic inducement, suggesting there may be an OSD bottleneck at a single-cell level. Furthermore, cells on scaffolds exhibited gradually reduced total migration rate (MR) and relatively constant local MR, both ideal for bone regeneration. Excellent adhesion of MSCs to the smooth scaffolds surface, especially the layer structures, was seen on scanning electron microscopy images, demonstrating fine compatibility between scaffold and cells. Our results indicate this simplified, integrated and potentially modularizable 3D bioreactor could enable the osteocytes producing from MSCs for expected applications.     

Analysis of β-galactosidase production and their genes of two strains of <Emphasis Type="Italic">Lactobacillus bulgaricus</Emphasis>

A bacterial β-galactosidase delivery system is a potential therapy for lactose intolerance. Currently, two Lactobacillus bulgaricus strains with different biological characteristics are under consideration as potential sources. However, differences in these β-galactosidase genes and their resulting production levels are poorly characterized. The β-galactosidase ORF of L. bulgaricus yogurt isolate had high variability and was terminated at site 1924 due to a stop codon. However, the full 114 kDa β-galactosidase band was still resolved by SDS-PAGE, which may indicate that the interrupted ORF was translated into more than one peptide, and they together were folded into the complete enzyme protein that showed much higher β-galactosidase activity (6.2 U/mg protein) than the enzyme generated from L. bulgaricus reference strain (2.5 U/mg protein).     

热量限制延缓人二倍体成纤维细胞衰老的体外模型

为建立人二倍体成纤维细胞IMR 90的热量限制体外模型 ,分别采用低浓度、正常浓度和高浓度葡萄糖培养条件 ,常规传代培养IMR 90细胞 ,利用综合细胞衰老指标对模型进行评价 .低、正常和高浓度葡萄糖培养条件组IMR 90细胞平均寿限分别为 5 8 3、5 5 0和 4 7 2PDL(群体倍增水平 ) .低浓度葡萄糖培养IMR 90细胞早期增长速度有所减慢 ,但仍保持对生长因子诱导的细胞增殖能力 ,并使晚期IMR 90处于细胞周期S期的比例以及其DNA修复能力显著高于其他条件培养的晚期细胞 .低浓度葡萄糖培养IMR 90晚期细胞的半乳糖苷酶染色阳性率亦明显低于其他条件培养的晚期细胞 .实验结果表明 ,低浓度葡萄糖培养可以延缓IMR 90复制衰老 ,建立了热量限制延缓衰老体外模型 ,为进一步探讨热量限制延缓衰老作用机制的研究打下基础     

Further observations on the effect of ethionine on the synthesis of β-galactosidase inEscherichia coli: Formation of an immunologically cross-reacting protein

It was found that ethionine partially inhibits the transport of the inducer (TMG) of β-galactosidase into the cells ofEscherichia coli ML-30. The synthesis of β-galactosidase-specific messenger RNA is not inhibited. Ethionine appears to be incorporated into proteins synthesized by the strains used. The incorporation of ethionine into the molecule of β-galactosidase results in the synthesis of an enzymically inactive, immunologically cross-reacting protein.     

β-Galactosidase production by the psychrotolerant yeast Guehomyces pullulans 17-1 isolated from sea sediment in Antarctica and lactose hydrolysis

A psychrotolerant yeast Guehomyces pullulans 17-1 was isolated from sea sediment in Antarctica. It was found that it could yield both extracellular and cell-bound β-galactosidase. After optimization of the medium and cultivation conditions, it was found that the yeast strain produced over 17.2 U mL⁻¹ of β-galactosidase activity within 120 h at the flask level while the yeast strain produced over 25.3 U mL⁻¹ of β-galactosidase activity within 144 h during the 2-L fermentation. This is the highest β-galactosidase activity produced by the wild type yeast strains reported so far. However, the optimal pH and temperature for the crude β-galactosidase were 4.0 and 50 °C, respectively. Lactose could be converted into glucose and galactose and a large amount of reducing sugar could be released from milk under catalysis of the yeast culture.     

A multiplex PCR technique to characterize human bone marrow derived mesenchymal stem cells

Human mesenchymal stem cells (MSCs), with capacity to differentiate into adipocytes, osteoblasts and chondrocytes, offer potential for the development of novel treatments. A critical question in MSCs biology is whether this cell population possesses a relatively uniform differentiation capability or is comprised of distinct subsets of progenitors committed to differentiate in particular pathways. To quantify the changes during growth of MSCs, we analyzed the mesenchymal phenotype and differentiation ability using a multi-marker PCR with six primer sets specific for CD73, CD90, CD105, CD166, CD45 and β-actin allowing a gel-based differential detection of the PCR products. To determine degree of variability of MSCs populations in terms of proliferation, cell proliferation assays were performed on expanded MSCs up to the sixth passage. At each passage, the osteogenic and adipogenic differentiation potentials of MSCs were verified by culture in inductive media. RT-PCR and cytochemical analysis revealed that, despite the loss of multipotentiality during expansion, certain markers remain expressed, indicating that these markers are unlikely to be reflective of the MSC’s true ‘stem cell’ nature. Our results suggest that decrease in the expression of MSCs specific markers correlates with down-regulation of proliferation ability and differentiation efficiency of MSCs.     

Growth and β-galactosidase synthesis in aerobic chemostat cultures of Kluyveromyces lactis

Growth and β-galactosidase (β-gal) expression were characterized in the yeast Kluyveromyces lactis strain NRRL Y-1118 growing in aerobic chemostat cultures under carbon, nitrogen or phosphate limitation. In lactose or galactose-limited cultures, β-gal accumulated in amounts equivalent to 10–12% of the total cell protein. The induced β-gal expression was repressed when cells were grown under N- or P-limitation. In lactose medium, enzyme levels were 4–8 times lower than those expressed in C-limited cultures. A similar response was observed when galactose was the carbon source. These results suggest that a galactose-dependent signal (in addition to glucose) may have limited induction when cells were grown in carbon-sufficient cultures. Constitutive β-gal expression was highest in lactate-limited and lowest in glucose-limited media and was also repressed in glucose-sufficient cultures. Other K. lactis strains (NRRL Y-1140 and CBS 2360) also showed glucose repression (although with different sensitivity) under non-inducing conditions. We infer that these strains share a common mechanism of glucose repression independent of the induction pathway. The kinetics of β-gal induction observed in C-limited cultures confirms that β-gal induction is a short-term enzyme adaptation process. Applying a lactose pulse to a lactose-limited chemostat culture resulted in ‘substrate-accelerated death’. Immediately after the pulse, growth was arrested and β-gal was progressively inactivated. Yeast metabolism in C-limited cultures was typically oxidative with the substrate being metabolized solely to biomass and CO₂. Cells grown under P- or N-limitation, either with glucose or lactose, exhibited higher rates of sugar consumption than C-limited cells, accumulated intracellular reserve carbohydrates and secreted metabolic products derived from the glycolytic pathway, mainly glycerol and ethanol. Received 16 October 1997/ Accepted in revised form 17 April 1998     

Multipotent mesenchymal stem cells of desquamated endometrium: Isolation,characterization, and application as a feeder layer for maintenance of human embryonic stem cells

In this study, we characterize new multipotent human mesenchymal stem cell lines (MSCs) derived from desquamated (shedding) endometrium of menstrual blood. The isolated endometrial MSC (eMSC) is an adhesive to plastic heterogeneous population composed mainly of endometrial glandular and stromal cells. The established cell lines meet the criteria of the International Society for Cellular Therapy for defining multipotent human MSCs of any origin. The eMSCs have positive expression of CD13, CD29, CD44, CD73, CD90, and CD105 markers and lack hematopoietic cell surface antigens CD19, CD34, CD45, CD117, CD130, and HLA-DR (class II). Multipotency of the established eMSCs is confirmed by their ability to differentiate into other mesodermal lineages, such as osteocytes and adipocytes. In addition, the isolated eMSCs partially (over 50%) express the pluripotency marker SSEA-4. However, they do not express Oct-4. Immunofluorescent analysis of the derived cells revealed the expression of the neural precursor markers nestin and β-III-tubulin. This suggests a neural predisposition of the established eMSCs. These cells are characterized by a high proliferation rate (doubling time 22–23 h) and a high colony-forming efficiency (about 60%). In vitro, the eMSCs undergo more than 45 population doublings without karyotypic abnormalities. We demonstrate that mitotically inactivated eMSCs are perfect feeder cells for maintenance of human embryonic stem cell lines (hESCs) C612 and C910. The eMSCs, being a feeder culture, sustain the hESC pluripotent status that verified by expression of Oct-4, alkaline phosphatase and SSEA-4 markers. The hESCs cocultured with the eMSCs retain their morphology and proliferative rate for more than 40 passages and exhibit the capability for spontaneous differentiation into embryoid bodies comprising three embryonic germ layers. Thus, an easy and noninvasive isolation of the eMSCs from menstrual blood, their multipotency and high proliferative activity in vitro without karyotypic abnormalities demonstrate the potential of use of these stem cells in regenerative medicine. Using the derived eMSCs as the feeder culture eliminates the risks associated with animal cells while transferring hESCs to clinical setting.     

Improvement of culture conditions to overproduce β-galactosidase from Escherichia coli in Bacillus subtilis

The effect of some culture variables in the production of β-galactosidase from Escherichia coli in Bacillus subtilis was evaluated. The lacZ gene was expressed in B. subtilis using the regulatory region of the subtilisin gene aprE. The host contained also the hpr2 and degU32 mutations, which are known to overexpress the aprE gene. We found that, when this overproducing B. subtilis strain was grown in mineral medium supplemented with glucose (MMG), β-galactosidase production was partially growth-associated, as 40%–60% of the maximum enzyme activity was produced before the onset of the stationary phase. In contrast, when a complex medium was used, β-galactosidase was produced only at low levels during vegetative growth, whereas it accumulated to high levels during early stationary phase. Compared with the results obtained in complex media, a 20% increase in specific β-galactosidase activity in MMG supplemented with 11.6 g/l glucose was obtained. On the 1-l fermenter scale, a threefold increase in volumetric β-galactosidase activity was obtained when the glucose concentration was varied from 11 g/l to 26 g/l. In addition, glucose feeding during the stationary phase resulted in a twofold increase in volumetric enzyme activity as cellular lysis was prevented. Finally, we showed that oxygen uptake and carbon dioxide evolution rates can be used for on-line determination of the onset of stationary phase, glucose depletion and biomass concentration. Received: 18 April 1996 / Received revision: 27 August 1996 / Accepted: 6 September 1996     

Isolation and characterization of novel murine epiphysis derived mesenchymal stem cells

Background

While bone marrow (BM) is a rich source of mesenchymal stem cells (MSCs), previous studies have shown that MSCs derived from mouse BM (BMMSCs) were difficult to manipulate as compared to MSCs derived from other species. The objective of this study was to find an alternative murine MSCs source that could provide sufficient MSCs.

Methodology/Principal Findings

In this study, we described a novel type of MSCs that migrates directly from the mouse epiphysis in culture. Epiphysis-derived MSCs (EMSCs) could be extensively expanded in plastic adherent culture, and they had a greater ability for clonogenic formation and cell proliferation than BMMSCs. Under specific induction conditions, EMSCs demonstrated multipotency through their ability to differentiate into adipocytes, osteocytes and chondrocytes. Immunophenotypic analysis demonstrated that EMSCs were positive for CD29, CD44, CD73, CD105, CD166, Sca-1 and SSEA-4, while negative for CD11b, CD31, CD34 and CD45. Notably, EMSCs did not express major histocompatibility complex class I (MHC I) or MHC II under our culture system. EMSCs also successfully suppressed the proliferation of splenocytes triggered by concanavalin A (Con A) or allogeneic splenocytes, and decreased the expression of IL-1, IL-6 and TNF-α in Con A-stimulated splenocytes suggesting their anti-inflammatory properties. Moreover, EMSCs enhanced fracture repair, ameliorated necrosis in ischemic skin flap, and improved blood perfusion in hindlimb ischemia in the in vivo experiments.

Conclusions/Significances

These results indicate that EMSCs, a new type of MSCs established by our simple isolation method, are a preferable alternative for mice MSCs due to their better growth and differentiation potentialities.     

Cloning and characterization of a β-galactosidase encoding region in Lactobacillus coryniformis CECT 5711

A chromosomal DNA fragment of 7.8 kb from Lactobacillus coryniformis CECT 5711 was cloned in Escherichia coli K-12 and was found to express a functional β-galactosidase. Nucleotide sequence analysis showed that this fragment contained two partially overlapping genes, the lacL (1,881 bp) and the lacM (960 bp), that encode the subunits of a heterodimeric β-galactosidase, with estimated molecular masses of 72,129 and 35,233 Da, respectively. Other three incomplete open reading frames showing homology to another β-galactosidase, an α-galactosidase, and a galactokinase, respectively, were also found. The L. coryniformis β-galactosidase was overproduced in E. coli by using an isopropyl-β-d-thiogalactopyranoside (IPTG) expression system. Two new proteins with an estimated M _r s of approximately 72,000 and 35,000 appeared upon induction with IPTG, and extracts of the recombinant E. coli strain showed β-galactosidase activity.     

Effect of ethionine on the synthesis of β-galactosidase inEscherichia coli

Ethionine at concentrations of 10⁻³M, 5×10⁻³M and 10⁻²M inhibits growth, both of β-galactosidase inducible ML-30 and constitutive ML-308Escherichia coli strains. The protein synthesis (measured by the incorporation of l-leucine-¹⁴C and l-aspartic-¹⁴C acid into proteins) of these strains is inhibited to the same extent as their growth. The synthesis of inducible and constitutive β-galactosidase produced by the strains ML-30 and ML-308, respectively, is considerably inhibited by ethionine.     

模拟微重力对骨髓间充质干细胞增殖及其向脂肪方向分化能力的影响

目的：探讨模拟微重力（SMG）对骨髓间充质干细胞（MSCs）的增殖及向脂肪方向分化能力的影响。方法：第一部分将第三代的MSCs分为两组,分别在正常重力下（NG组）及微重力下（SMG组,采用回转模拟装置以30r/min回转模拟微重力）,培养72h后,采用BrdU标记法检测两组细胞的增殖情况,细胞计数法绘制细胞生长曲线。Western Blot检测干细胞标志物Oct4、SSEA4的表达情况,第二部分将第三代MSCs分为三组：第一组在NG条件下培养后,加入脂肪方向诱导剂在NG条件下诱导、第二组在SMG条件下培养,在NG条件下诱导,第三组在SMG条件下培养,在SMG条件下诱导。7天后,油红O染色观察脂肪方向的诱导率,Western Blot检测过氧化物酶增殖物激活受体γ2（PPARγ2）以及Oct4的表达。结果：第一部分：流式细胞仪检测SMG组BrdU标记阳性率明显高于NG组,表明细胞增殖较快,Western Blot结果显示SMG组细胞中Oct4、SSEA4的表达量明显高于NG组,有统计学意义。第二部分：脂肪方向诱导后第一组细胞油红O染色阳性,Western Blot显示PPARγ2呈阳性表达,Oct4仅有微量表达,第二组油红O染色阳性表达率明显高于第一组,且PPARγ2表达较第一组增多,几乎未见Oct4的表达,第三组细胞油红O染色阴性,且几乎不表达PPARγ2,而Oct4表达较前两组升高。结论：模拟微重力可促进骨髓间充质干细胞增殖,提高其向脂肪方向分化的能力可能与微重力保持其未分化状态相关。     

Effect of 5-azacytidine on the protein expression of porcine bone marrow mesenchymal stem cells in vitro

Bone marrow-derived mesenchymal stem cells （MSCs） are pluripotent stem cells that show a vital potential in the clinical application for cell transplantation. In the present paper, proteomic techniques were used to approach the protein profiles associated with porcine bone marrow MSCs and investigate the regulation of MSC proteins on the effect of 5-azacytidine （5-aza）. Over 1,700 protein species were separated from MSCs according to gel analysis. Compared with the expression profiling of control MSCs, there were 11 protein spots up-regulated and 26 downregulated in the protein pattern of 5-aza-treated cells. A total of 21 proteins were successfully identified by MALDI-TOF-MS analysis, among which some interesting proteins, such as alpha B-crystallin, annexin A2, and stathmin 1, had been reported to involve in cell proliferation and differentiation through different signaling pathways. Our data should be useful for the future study of MSC differentiation and apoptosis.     

A dose-dependent function of follicular fluid on the proliferation and differentiation of umbilical cord mesenchymal stem cells (MSCs) of goat

Umbilical cord (UC) has been suggested as a new source of mesenchymal stem cells (MSCs). In this report, we isolated MSCs from the fetal UC of goat and investigated their multipotency of differentiation into germ cells in vitro, in the presence of 0-20?% bovine follicular fluid (FF). The phenotypes, capacity of proliferation and expression of MSC markers were served as the indexes of multipotency of the isolated UC-MSCs, those were ascertained by growth curves, RT-PCR and immunofluorescent staining, respectively. Our results showed that the UC-MSCs shared a similar immunophenotype to those cells reported in mouse and human bone marrow MSCs, as well as some characteristics seen in embryonic stem cells (ESCs). In addition, our data also demonstrated that a dose-dependent function of FF on the states of differentiation of goat UC-MSCs. From 2 to 20?% of the FF can promote the proliferation of goat UC-MSC, especially the 5?% concentration of follicular fluid promote proliferation was significantly higher than 2?%. In contrast, higher concentration of follicular fluid (>10?%) induced goat UC-MSCs differentiation into oocyte-like cells. These findings provide an efficient model to study the mechanism on cell proliferation and germ cell differentiation in livestock using FF.