Effects of products from macrophages, blood mononuclear cells and of retinol on collagenase secretion and collagen synthesis in chondrocyte culture

Collagenase secretion was studied in cultures of rabbit articular chondrocytes. Differentiation of the cells was assessed by characterizing the type of ³H-labelled collagen produced during treatment with (1) conditioned media from rabbit peritoneal macrophages and human blood mononuclear cells, and (2) with retinol, a potent cartilage resorbing agent in tissue culture. Conditioned media stimulated collagenase secretion. Total collagen synthesis was reduced due to a decrease of synthesis of α₁ chains; the amount of α₂ chains synthesized was unchanged. This is thought to be due to a reduction in type II synthesis. Retinol did not stimulate collagenase secretion. Total collagen synthesis was reduced by retinol. α₂ chain synthesis, however, was significantly increased, suggesting a switch of collagen synthesis in favor of type I collagen and, therefore, dedifferentiation. These results demonstrate that dedifferentiation of chondrocytes with respect to collagen synthesis is not necessarily associated with a stimulation of collagenase secretion.     

Galectin-1 in cartilage: Expression, influence on chondrocyte growth and interaction with ECM components

Galectin-1 is a 14 kDa beta-galactoside binding protein, capable of forming lattice-like structures with glycans of cellular glycoconjugates and inducing intracellular signaling. The expression of Galectin-1 in porcine cartilage is described in this work for the first time. Immunocytochemical methods revealed distinct distribution patterns for both articular and growth plate cartilage. In articular cartilage, the highest reactivity for Galectin-1 was found in all chondrocytes at the superficial zone and in most of those at the lower layer of the middle zone. In the growth plate, marked reactivity was seen in chondrocytes at the proliferative zone and reached a maximum level for the column-forming cells at the hypertrophic zone. In addition, different Galectin-1 distribution patterns were observed at the subcellular level. With regards to the metabolic effects of Galectin-1, the results in vitro seem to indicate an inhibitory effect of Galectin-1 on articular chondrocyte anabolism (i.e. inhibition of cell proliferation and anabolic gene expression) and a stimulation of catabolic processes (i.e. induction of matrix degradation and hypertrophy marker expression). These data represent a starting point for the understanding the molecular mechanisms underlining ECM-Galectin-1 interaction and the subsequent signaling-cell transduction processes involving cartilage formation and maturation.     

The effect of ascorbate on embryonic chick sternal chondrocytes cultured in agarose

Primary cultures of embryonic chick sternal chondrocytes were embedded in a three-dimensional matrix of 1% solid agarose which was overlaid with nutrient media. The chondrocytes divided and formed nests of spherically shaped cells which were surrounded by an extensive extracellular matrix containing high molecular weight proteoglycans. Using light and electron microscopy, condensation of proteoglycan was observed pericellularly, often forming septa between cells of a nest, and as part of the outer boundary of the cell nest. No cross-striated collagen fibers were observed in the extracellular matrix although proteoglycan appeared to decorate a network of fine strands. Upon the addition of ascorbate to the nutrient media high molecular weight proteoglycans were synthesized, but there was a marked decrease in the synthesis of proteoglycans after a 10 day exposure to ascorbate. Morphologically, the decrease in proteoglycan synthesis was manifested in the discontinuous arrangement of the pericellular matrix as well as the diffuse form of the cell-nest boundary. Both of these structures were clearly defined in control cultures and were enriched in proteoglycan as demonstrated by ruthenium red staining. This study demonstrates that embryonic chondrocytes remain differentiated when cultured in solid agarose for a period of up to 15 days. They continue to synthesize their tissue specific macromolecules and are phenotypically stable when exposed to ascorbate for extended periods of time.     

Multiple activation of mitogen-activated protein kinases by purified independent CCN2 modules in vascular endothelial cells and chondrocytes in culture

CCN2 consists of 4 distinct modules that are conserved among various CCN family protein members. From the N-terminus, insulin-like growth factor binding protein (IGFBP), von Willebrand factor type C repeat (VWC), thrombospondin type 1 repeat (TSP1) and C-terminal cysteine-knot (CT) modules are all aligned tandem therein. The multiple functionality of CCN2 is thought to be enabled by the differential use of these modules when interacting with other molecules. In this study, we independently prepared all 4 purified module proteins of human CCN2, utilizing a secretory production system with Brevibacillus choshinensis and thus evaluated the cell biological effects of such single modules. In human umbilical vascular endothelial cells (HUVECs), VWC, TSP and CT modules, as well as a full-length CCN2, were capable of efficiently activating the ERK signal transduction cascade, whereas IGFBP was not. In contrast, the IGFBP module was found to prominently activate JNK in human chondrocytic HCS-2/8 cells, while the others showed similar effects at lower levels. In addition, ERK1/2 was modestly, but significantly activated by IGFBP and VWC in those cells. No single module, but a mixture of the 4 modules provoked a significant activation of p38 MAPK in HCS-2/8 cells, which was activated by the full-length CCN2. Therefore, the signals emitted by CCN2 can be highly differential, depending upon the cell types, which are thus enabled by the tetramodular structure. Furthermore, the cell biological effects of each module on these cells were also evaluated to clarify the relationship among the modules, the signaling pathways and biological outcomes. Our present results not only demonstrate that single CCN2 modules were potent activators of the intracellular signaling cascade to yield a biological response per se, while also providing new insight into the module-wise structural and functional relationship of a prototypic CCN family member, CCN2.     

Adult human arterial smooth muscle cells in primary culture Modulation from contractile to synthetic phenotype

Summary Smooth muscle cells were isolated enzymatically from adult human arteries, grown in primary culture in medium containing 10% whole blood serum, and studied by transmission electron microscopy and [³H]thymidine autoradiography. In the intact arterial wall and directly after isolation, each smooth muscle cell had a nucleus with a wide peripheral zone of condensed chromatin and a cytoplasm dominated by myofilament bundles with associated dense bodies. After 1–2 days of culture, the cells had attached to the substrate and started to spread out. At the same time, a characteristic fine-structural modification took place. It included nuclear enlargement, dispersion of the chromatin and formation of large nucleoli. Moreover, myofilament bundles disappeared and an extensive rough endoplasmic reticulum and a large Golgi complex were organized in the cytoplasm. This morphological transformation of the cells was completed in 3–4 days. It was accompanied by initiation of DNA replication and mitosis.The observations demonstrate that adult human arterial smooth muscle cells, when cultivated in vitro, pass through a phenotypic modulation of the same type as arterial smooth muscle cells from experimental animals. This modulation gives the cells morphological and functional properties resembling those of the modified smooth muscle cells found in fibroproliferative lesions of atherosclerosis. Further studies of the regulation of smooth muscle phenotype and growth may provide important clues for a better understanding of the pathogenesis of atherosclerosis.     

Phenotypic and AFLP characterization of two new pineapple somaclones derived from in vitro culture

Fifty pineapple buds (cv. Red Spanish Pinar, donor) were collected from field-grown plants and cultured in vitro. Forty-three young pineapple shoots were obtained after 42 d of implantation. Shoots were micropropagated for 168 d to produce 24,768 shoots. Three hundred young leaves were randomly selected as explants for callus formation. Calli proliferated for 4 months. Five hundred calli were randomly selected and transferred to the plantlet regeneration medium. Four hundred twenty-seven in vitro-plantlets were obtained and later hardened ex vitro. Then, 387 plantlets were transferred to the field environment and asexually propagated for two generations (30 months). Only two phenotype variants were identified: P3R5 and Dwarf. A more detailed study was carried out to compare these two variants with the donor plant. The variant P3R5 showed differences in the number of slips and suckers, and in the presence of thorns in the leaves and in the fruit crowns. The somaclonal variant Dwarf, was different from the donor plant in regard with the plant height; the peduncle diameter; the number of shoots, slips and suckers; the fruit mass with crown; the number of eyes in the fruit; the fruit height and diameter; the leaf color; the plant architecture; the length of plant generation cycle; and the fruit color and shape. Both somaclonal variants showed different AFLP banding patterns in comparison with the donor cultivar.     

Enhanced nerve growth factor efficiency in neural cell culture by immobilization on the culture substrate

Nerve growth factor (NGF) immobilization on a culture substrate may dramatically reduce the amount of NGF required for pheochromocytoma (PC12) cell culture. Coverslips on which NGF had been immobilized, or with NGF added to the culture medium daily, were used to culture PC12 cells. We examined the effects of adding 5, 10, or 100 ng of NGF to cultures daily, and compared them to the effects of immobilizing 5, 10, or 100 ng of NGF on culture substrates in a single dose. Cultures with 10 or 5 ng NGF added daily showed dramatically decreased cell viability, mitochondrial metabolic activity, and neuronal differentiation compared to cultures with 100 ng NGF added daily, while also exhibiting increased apoptosis. In contrast, a single dose of 100 ng immobilized NGF yielded results similar to 100 ng NGF added daily (total: 300 ng over 3 days), and 10 or 5 ng immobilized NGF showed far better results than 10 or 5 ng NGF added daily. These results demonstrate that NGF immobilization can dramatically reduce the amount of NGF required in neuronal cell culture.     

Effects of growth rate and influent substrate concentration on effluent quality from chemostats containing bacteria in pure and mixed culture

Studies were performed using pure cultures of A. acrogenes and E. coli and a heterogeneous microbial population growing in carbon-limited chemostats with glucose as the sole carbon and energy source. A two-level factorial experimental design was employed to test the hypothesis that the concentration of growth-limiting substrate in a chemostat is controlled by the growth rate alone and is independent of the concentration of substrate entering the reactor. The pure culture experiments showed that the conclusions depend upon the measurement employed for growth-limiting substrate. When the concentration of glucose was measured directly, the hypothesis was found to be true within the limits of the study (500–1500 mg/liter). However, if the chemical oxygen demand (COD) test was used as the measure of growth-limiting substrate the hypothesis was found to be false. When heterogeneous cultures were employed the hypothesis was false regardless of the technique used to measure the concentration of growth-limiting substrate. Nevertheless, it was possible to generate regression equations which described the interactions among influent COD, growth rate, and effluent COD with a high level of correlation.     

A conceptual review on systems biology in health and diseases: from biological networks to modern therapeutics

Human physiology is an ensemble of various biological processes spanning from intracellular molecular interactions to the whole body phenotypic response. Systems biology endures to decipher these multi-scale biological networks and bridge the link between genotype to phenotype. The structure and dynamic properties of these networks are responsible for controlling and deciding the phenotypic state of a cell. Several cells and various tissues coordinate together to generate an organ level response which further regulates the ultimate physiological state. The overall network embeds a hierarchical regulatory structure, which when unusually perturbed can lead to undesirable physiological state termed as disease. Here, we treat a disease diagnosis problem analogous to a fault diagnosis problem in engineering systems. Accordingly we review the application of engineering methodologies to address human diseases from systems biological perspective. The review highlights potential networks and modeling approaches used for analyzing human diseases. The application of such analysis is illustrated in the case of cancer and diabetes. We put forth a concept of cell-to-human framework comprising of five modules (data mining, networking, modeling, experimental and validation) for addressing human physiology and diseases based on a paradigm of system level analysis. The review overtly emphasizes on the importance of multi-scale biological networks and subsequent modeling and analysis for drug target identification and designing efficient therapies.     

The effect of substrate and epidermal growth factor on human placental trophoblast cells in culture

Summary Attempts were made to select for trophoblast cells in cultures of mixed cell populations derived from preterm (7 to 12 wk) or term human placentas. Epidermal growth factor added to cultures on solid or porous supports caused proliferation of epithelial-type cells to give a confluent monolayer but did not increase the expression of differentiated function. The presence or absence of placental basement membrane collagen as substrate made little apparent difference; however a porous basement membrane collagen support led to increased differentiated function. Initial production of human chorionic gonadotrophin was increased and after 4 wk in culture a substantial proportion of the cells exhibited alkaline phosphatase activity. Epidermal growth factor and a substrate of placental basement membrane collagen on a porous support favorably influence the growth and differentiation of human trophoblast cells in culture. This work was supported by funds from the Medical Research Council of New Zealand which also provided support for Dr. Truman as a Postdoctoral Fellow.     

Effect of formaldehyde on growth of obligate methylotroph Methylobacillus flagellatum in a substrate non-limited continuous culture

The ribulose monophosphate cycle methylotroph Methylobacillus flagellatum was grown under oxyturbidostat conditions on mixtures of methanol and formaldehyde. Formaldehyde when added at low concentration (50 mg/l) increased the methanol consumption and the yield of biomass. The presence of 150–300 mg/l of formaldehyde resulted in an increase of the growth rate from 0.74 to about 0.79–0.82 h^-1. The presence of 500 mg/l of formaldehyde in the inflow decreased culture growth characteristics. Activities of methanol dehydrogenase and enzymes participating in formaldehyde oxidation and assimilation were measured. The enzymological profiles obtained are discussed.Abbreviations MDH methanol dehydrogenase - NAD-linked FDDH NAD-linked formaldehyde dehydrogenase - DLFDDH dye-linked formaldehyde dehydrogenase - DLFDH dye-linked formate dehydrogenase - GPDH glucose-6-phosphate dehydrogenase - PGDH 6-phosphogluconate dehydrogenase - RuMP cycle ribulose monophosphate cycle     

Application of solid waste from anaerobic digestion of poultry litter in Agrocybe aegerita cultivation: mushroom production, lignocellulolytic enzymes activity and substrate utilization

The degradation and utilization of solid waste (SW) from anaerobic digestion of poultry litter by Agrocybe aegerita was evaluated through mushroom production, loss of organic matter (LOM), lignocellulolytic enzymes activity, lignocellulose degradation and mushroom nutrients content. Among the substrate combinations (SCs) tested, substrates composed of 10–20% SW, 70–80% wheat straw and 10% millet was found to produce the highest mushroom yield (770.5 and 642.9 g per 1.5 kg of substrate). LOM in all SCs tested varied between 8.8 and 48.2%. A. aegerita appears to degrade macromolecule components (0.6–21.8% lignin, 33.1–55.2% cellulose and 14–53.9% hemicellulose) during cultivation on the different SCs. Among the seven extracellular enzymes monitored, laccase, peroxidase and CMCase activities were higher before fruiting; while xylanase showed higher activities after fruiting. A source of carbohydrates (e.g., millet) in the substrate is needed in order to obtain yield and biological efficiency comparable to other commercially cultivated exotic mushrooms.     

Effects of retinol and dexamethasone on cytokine-mediated control of metalloproteinases and their inhibitors by human articular chondrocytes and synovial cells in culture

Human articular chondrocytes in culture produced large amounts of specific mammalian collagenase, gelatinase and proteoglycanase when exposed to dialysed supernatant medium derived from cultured human blood mononuclear cells (mononuclear cell factor) or to conditioned medium, partially purified by fractionation with ammonium sulphate (60–90% fraction), from cultures of human synovial tissue (synovial factor). Human chondrocytes and synovial cells also released into culture medium an inhibitor of collagenase of apparent molecular weight about 30 000, which appeared to be similar to the tissue inhibitor of metalloproteinases synthesised by tissues in culture. The amounts of free collagenase inhibitor were reduced in culture media from chondrocytes or synovial cells exposed to mononuclear cell factor or synovial factor. While retinol inhibited the production of collagenase brought about by mononuclear cell factor or synovial factor, it restored the levels of inhibitor, which were reduced in the presence of mononuclear cell factor or synovial factor. Dexamethasone markedly reduced the production of collagenase by synovial cells, while only partially inhibiting factor-stimulated collagenase production by chondrocytes. Addition of puromycin as an inhibitor of protein synthesis reduced the amounts of both collagenase and inhibitor to control or undetectable levels.     

Fermentative production of P(3HB-co-3HV) from propionic acid by Alcaligenes eutrophus in fed-batch culture with pH-stat continuous substrate feeding method

The feeding of propionic acid for production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] by Alcaligenes eutrophus ATCC17697 was optimized using a fed-batch culture system. The concentration of propionic acid was maintained at 3 g l^–1 as growth was inhibited by propionic acid in the broth. A pH-stat substrate feeding system was used in which propionic acid was fed automatically to maintain a pH of the culture broth at 7.0. By feeding a substrate solution containing 20% (w/v) propionic acid, 4.9% (w/v) ammonia water [at a molar ratio of carbon to nitrogen (C/N molar ratio) of 10] in cell growth phase, the concentration of propionic acid in the broth was maintained at 3 g l^–1 giving a specific growth rate of 0.4 h^–1. To promote P(3HB-co-3HV) production, two stage fed-batch culture which consisted of the stage for the cell growth and the stage for the P(3HB-co-3HV) accumulation was carried out. When the substrate solution whose C/N molar ratio was 50 was fed in P(3HB-co-3HV) accumulation phase, the cell concentration and the P(3HB-co-3HV) content in the cells reached 64 g l^–1 and 58% (w/w) in 55.5 h, respectively.     

Effect of peripheral nerve on the neurite growth from retinal explants in culture

The effect of peripheral nerve (PN) on neurite outgrowth from retinal explants of adult hamsters was examined.Cultures of retinal explants,and co-cultures of retinal explants and PN were performed using chick retinal basement memebrane (BM) as substrate.The presence of PN increases the number and length of neurite outgrowth.In addition,a high proportion of neurites situated close to PN tend to grow towards it.Since there was no contact between retinal explants and PN,we suggest that PN might secete diffusible substances to attract the neurites to grow towards it.     

Influence of sample preparation on cellular glutathione recovery from adherent cells in culture

During the last decade, the unbound glutathione content of cultured adherent cells has become a very important biological marker for many pharmacological and toxicologicalin vitro studies with regard to the protective role of the tripeptide in its reduced form (GSH). However, the literature does not provide extensive information on the influence of sample preparation on cellular GSH and thiol analyses. Using the fibroblast-like V79 cell line as model, we undertook a comparative study of the efficiency of different procedures reported in the literature with respect to GSH recovery. Depending on the preanalytical step, up to 10-fold discrepancies could be observed in the recovery of intracellular GSH. Different parameters that must be controlled in order to maximize GSH recovery are discussed. The optimal strategy consisted in rapid perchloric acid deproteinization performed directly in the dish, which was extremely valuable for preparing GSH samples from adherent cells, and especially from cells expressing elevated -glutamyl transferase activity.Abbreviations EDTA ethylenediaminetetraacetic acid - GGT -glytamyl transferase (EC 2.3.2.2) - GSH reduced glutathione - HPLC high-performance liquid chromatography - PA perchloric acid - PBS Dulbecco's phosphate-buffered saline     

小鼠2,4,8—细胞胚胎卵裂球体外培养的研究

以ＣＺＢ为基础培养液,培养小鼠２、４、８－细胞胚胎的卵裂球,研究葡萄糖、牛磺酸和胰岛素对１／２卵裂球体外发育的影响及１／４、１／８和２／８卵裂球的体外发育规律。２－细胞胚胎卵裂球在ＣＺＢ中和在添加牛磺酸的ＣＺＢ中培养,其囊胚发育率（分别为９２％、８９％）无显差异（Ｐ＞０．０５）。胰岛素在少量葡萄糖存在的情况下,不影响卵裂球的囊胚发育率;在无葡萄糖时,卵裂球的囊胚发育率（３８％）显降低。牛磺酸在