A perfusion air-lift bioreactor for high density plant cell cultivation and secreted protein production

A new bioreactor design that allows continuous perfusion cultivation of plant cell suspensions is described in this paper. This design incorporates an internal cell settling zone with an external-loop air-lift bioreactor. The settling zone is created by inserting a baffle plate into the upper portion of the downcomer. Using this bioreactor, Anchusa officinalis suspension culture was cultivated to a cell density of 27.2 g l⁻¹ DW in 14 days at a perfusion rate of 0.123 per day. The maximum total extracellular protein concentration attained 1.11 g l⁻¹. Complete cell retention was achieved throughout the culture during which the maximum packed cell volume (PCV) exceeded 80%. In comparison, the maximum cell density and extracellular protein concentration in the batch culture were 12.6 g l⁻¹ DW and 0.47 g l⁻¹, respectively. SDS-PAGE of the extracellular protein samples revealed two major bands at 58 and 47 kDa, each accounted for approximately 45% of the total secreted proteins.     

VEGF couples hypertrophic cartilage remodeling, ossification and angiogenesis during endochondral bone formation.

Hypertrophic chondrocytes in the epiphyseal growth plate express the angiogenic protein vascular endothelial growth factor (VEGF). To determine the role of VEGF in endochondral bone formation, we inactivated this factor through the systemic administration of a soluble receptor chimeric protein (Flt-(1-3)-IgG) to 24-day-old mice. Blood vessel invasion was almost completely suppressed, concomitant with impaired trabecular bone formation and expansion of hypertrophic chondrocyte zone. Recruitment and/or differentiation of chondroclasts, which express gelatinase B/matrix metalloproteinase-9, and resorption of terminal chondrocytes decreased. Although proliferation, differentiation and maturation of chondrocytes were apparently normal, resorption was inhibited. Cessation of the anti-VEGF treatment was followed by capillary invasion, restoration of bone growth, resorption of the hypertrophic cartilage and normalization of the growth plate architecture. These findings indicate that VEGF-mediated capillary invasion is an essential signal that regulates growth plate morphogenesis and triggers cartilage remodeling. Thus, VEGF is an essential coordinator of chondrocyte death, chondroclast function, extracellular matrix remodeling, angiogenesis and bone formation in the growth plate.     

An autoradiographic study of prostaglandin E1 and/or its metabolites in mouse kidney tissue

The use of grain density autoradiography was evaluated as a means of quantifying the distribution of injected prostaglandin E₁ and/or its metabolites in the mouse kidney. Fifty microcuries of ³H-PGE₁ were injected into each of three mice which were sacrificed at 20 min, 40 min and 45 h after injection. Sections of frozen kidneys were mounted on prepared liquid emulsion slides. Following appropriate exposure and processing, the radioactivity in various regions was quantified by grain counting using an eyepiece grid superimposed over the field at 1000X. The highest grain density was found over the papilla and the second highest grain density occurred at the region of the inner zone of the cortex. In addition, the percentage decrease of grain density from tissue taken 40 min after injection when compared to that from tissue taken 20 min after injection implied that these two regions retained the radioactivity more so than the outer zone of the cortex, the outer zone of the medulla and the glomeruli.     

c-Src-mediated Phosphorylation of Thyroid Hormone Receptor-interacting Protein 6 (TRIP6) Promotes Osteoclast Sealing Zone Formation

Osteoclasts resorb bone through the formation of a unique attachment structure called the sealing zone. In this study, a role for thyroid hormone receptor-interacting protein 6 (TRIP6) in sealing zone formation and osteoclast activity was examined. TRIP6 was shown to reside in the sealing zone through its association with tropomyosin 4, an actin-binding protein that regulates sealing dimensions and bone resorptive capacity. Suppression of TRIP6 in mature osteoclasts by RNA interference altered sealing zone dimensions and inhibited bone resorption, whereas overexpression of TRIP6 increased the sealing zone perimeter and enhanced bone resorption. Treatment of osteoclasts with lysophosphatidic acid (LPA), which phosphorylates TRIP6 at tyrosine 55 through a c-Src-dependent mechanism, caused increased association of TRIP6 with the sealing zone, as did overexpression of a TRIP6 cDNA bearing a phosphomimetic mutation at tyrosine 55. Further, LPA treatment caused increases in osteoclast fusion, sealing zone perimeter, and bone resorptive capacity. In contrast, overexpression of TRIP6 containing a nonphosphorylatable amino acid residue at position 55 severely diminished sealing zone formation and bone resorption and suppressed the effects of LPA on the cytoskeleton. LPA effects were mediated through its receptor isoform LPA(2), as indicated by treatments with receptor-specific agonists and antagonists. Thus, these studies suggest that TRIP6 is a critical downstream regulator of c-Src signaling and that its phosphorylation is permissive for its presence in the sealing zone where it plays a positive role in osteoclast bone resorptive capacity.     

Osteoclast and its roles in calcium metabolism and bone development and remodeling

Osteoclasts are multinucleated cells responsible for bone resorption and play important roles in normal skeletal development, in the maintenance of its integrity throughout life, and in calcium metabolism. During bone resorption, the cytoskeleton of osteoclasts undergoes extensive reorganization, with polarization and formation of ruffled borders to secrete acid and formation of sealing zone to prevent leakage. The differentiation and function of osteoclasts are in turn regulated by osteoblasts, stromal cells, and bone. They are also subjected to negative feedback regulation by extracellular and intracellular calcium concentrations.     

Expression of cathepsin K mRNA and protein in odontoclasts after experimental tooth movement in the mouse maxilla by in situ hybridization and immunoelectron microscopy

This study demonstrated the simultaneous expression of cathepsin K (CK) mRNA by in situ hybridization and CK protein by immunoelectron microscopy in odontoclasts in mouse maxillae after experimental tooth movement. On the pressure side (the area under pressure during tooth movement), CK mRNA was detected in odontoclasts in resorption lacunae in the tooth root, in osteoclasts in bone resorption lacuane, and in fibroblasts in the periodontal ligament. Using electron microscopy, CK protein was detected at the apex of odontoclasts, intracellularly in vesicles and granules, and extracellularly in irregularly shaped vacuoles (extracellular spaces), on the plasma membrane of the ruffled border, and on and between typical striated type I collagen fibrils in the lacunae. These vesicles and granules appeared to fuse with irregular vacuoles containing CK-positive fragmented fibril-like structures close to the ruffled border. In the basolateral portion of odontoclasts, small amounts of CK-positive rough endoplasmic reticulum (ER) were found. CK-positive intracellular vacuoles (not extracellular spaces) also appeared to fuse with the vesicles and granules. However, these fused organelles rarely contained fragmented fibril-like structures. They are probably endolysosomes. The distribution of CK in odontoclasts was similar to that previously seen in osteoclasts. Furthermore, CK-positive fibril-like structures were found in the vacuoles of fibroblasts. These results indicated that during tooth movement CK is synthesized in odontoclasts on the pressure side and secreted into the tooth resorption lacunae. Therefore, CK may take part in the degradation of the dentin matrix (type I collagen fibrils and non-collagenous protein) of the tooth root, and in the subsequent intracellular degradation of endocytosed fragmented fibril-like structures in endolysosomes.     

Podosome organization drives osteoclast-mediated bone resorption

Osteoclasts are the cells responsible for physiological bone resorption. A specific organization of their most prominent cytoskeletal structures, podosomes, is crucial for the degradation of mineralized bone matrix. Each podosome is constituted of an F-actin-enriched central core surrounded by a loose F-actin network, called the podosome cloud. In addition to intrinsic actin dynamics, podosomes are defined by their adhesion to the extracellular matrix, mainly via core-linking CD44 and cloud-linking integrins. These properties allow podosomes to collectively evolve into different patterns implicated in migration and bone resorption. Indeed, to resorb bone, osteoclasts polarize, actively secrete protons, and proteases into the resorption pit where these molecules are confined by a podosome-containing sealing zone. Here, we review recent advancements on podosome structure and regulatory pathways in osteoclasts. We also discuss the distinct functions of different podosome patterns during the lifespan of a single osteoclast.     

Defective microtubule-dependent podosome organization in osteoclasts leads to increased bone density in Pyk2(-/-) mice

The protein tyrosine kinase Pyk2 is highly expressed in osteoclasts, where it is primarily localized in podosomes. Deletion of Pyk2 in mice leads to mild osteopetrosis due to impairment in osteoclast function. Pyk2-null osteoclasts were unable to transform podosome clusters into a podosome belt at the cell periphery; instead of a sealing zone only small actin rings were formed, resulting in impaired bone resorption. Furthermore, in Pyk2-null osteoclasts, Rho activity was enhanced while microtubule acetylation and stability were significantly reduced. Rescue experiments by ectopic expression of wild-type or a variety of Pyk2 mutants in osteoclasts from Pyk2(-/-) mice have shown that the FAT domain of Pyk2 is essential for podosome belt and sealing zone formation as well as for bone resorption. These experiments underscore an important role of Pyk2 in microtubule-dependent podosome organization, bone resorption, and other osteoclast functions.     

Protein tyrosine kinase inhibitors increase cytosolic calcium and inhibit actin organization as resorbing activity in rat osteoclasts

Although there is evidence that protein tyrosine kinase inhibitors (PTKIs) suppress bone resorption activity, the mechanism of action of these compounds on osteoclastic bone resorption remains obscure. In the present study, we investigated the effect of PTKIs on cytosolic Ca(2+) concentration ([Ca(2+)](i)) and on the cytoskeleton in rat osteoclasts. The PTKIs, genistein and herbimycin A, reversibly elevated [Ca(2+)](i) measured by fura-2 microfluorimetry. The PTKI-induced increase was abolished by omission of extracellular Ca(2+), but was not attenuated by depletion of Ca(2+) stores. The PTKI-induced increase was inhibited by addition of La(3+) and Ni(2+), but not abolished by dihydropyridine (DHP) Ca(2+) channel blockers. Genistin, an inactive analogue of genistein, had no effect on [Ca(2+)](i). In the cytoskeleton assay, genistein rapidly disrupted the actin ring formation that serves as a marker for the resorbing state of osteoclasts. Disruption of the actin ring formation was also diminished in Ca(2+)-free extracellular solution. These results suggest that PTKIs in rat osteoclasts elevate [Ca(2+)](i) via activation of a DHP-insensitive, nonspecific Ca(2+) entry pathway and disrupt the formation of actin rings, resulting in suppression of bone resorption activity. The regulation of this Ca(2+)-influx by PTKIs is likely to contribute to inhibition of bone resorption by these compounds.     

Ribonucleic acid within the extracellular matrix during embryonic tooth formation

During embryonic tooth formation, interactions between epithelial and mesenchymal cells results in the formation of a metachromatic interface or extracellular matrix. The cervical or germinative region of this epidermal organ system is populated by an increasing gradient of cellular differentiation and an extracellular matrix which is the progenitor for subsequent dentine organic matrix formation. Embryonic rabbit tooth primordia can be maintained in culture enabling kinetic studies of labeled precursor incorporation. Autoradiographs of tooth organ cultures continusly incubated with labeled uridine for periods up to eight hours, demonstrated initial cellular incorporation with subsequent transfer of 2% of the grain density to the extracellular matrix by four hours. The grain density was removed by ribonuclease treatment. No incorporation of tritiated thymidine into the matrix was observed. The incorporation of C¹⁴-uridine during organ culture was inhibited by actinomycin D. Micrurgy was employed to isolate the extracellular matrix free of adherent cells. Electron microscopy demonstrated membrane-bound, electron dense bodies within the matrix, presumably cytoplasmic extensions. No cells per se were observed on the isolated matrix. Several experimental criteria suggested that uridine incorporation into the extracellular matrix was regulated by epithelial and mesenchymal cells. Phenol extraction procedures of labeled cervical matrices demonstrated an ultraviolet absorption maximum at 260 μU. Both spectrophotometric determinations and orcinol assays found RNA to be 0.4–0.5% of the cervical extracellular matrix. These results are interpreted to indicate that RNA is a component of the metachromatic extracellular matrix during epithelio-mensenchymal interactions associated with tooth formation. The functional significance of these observations is premature at this time.     

Fibroblast activation protein alpha is expressed by chondrocytes following a pro-inflammatory stimulus and is elevated in osteoarthritis

Arthritis is characterised by the proteolytic degradation of articular cartilage leading to a loss of joint function. Articular cartilage is composed of an extracellular matrix of proteoglycans and collagens. We have previously shown that serine proteinases are involved in the activation cascades leading to cartilage collagen degradation. The aim of this study was to use an active-site probe, biotinylated fluorophosphonate, to identify active serine proteinases present on the chondrocyte membrane after stimulation with the pro-inflammatory cytokines IL-1 and oncostatin M (OSM), agents that promote cartilage resorption. Fibroblast activation protein alpha (FAPalpha), a type II integral membrane serine proteinase, was identified on chondrocyte membranes stimulated with IL-1 and OSM. Real-time PCR analysis shows that FAPalpha gene expression is up-regulated by this cytokine combination in both isolated chondrocytes and cartilage explant cultures and is significantly higher in cartilage from OA patients compared to phenotypically normal articular cartilage. Immunohistochemistry analysis shows FAPalpha expression on chondrocytes in the superficial zone of OA cartilage tissues. This is the first report demonstrating the expression of active FAPalpha on the chondrocyte membrane and elevated levels in cartilage from OA patients. Its cell surface location and expression profile suggest that it may have an important pathological role in the cartilage turnover prevalent in arthritic diseases.     

Electron microscopic study of condylar cartilage of rat mandible stained with ruthenium red: proteoglycans and hypertrophic chondrocytes

Ultrastructure of hypertrophic chondrocytes and extracellular matrix in condylar cartilage of rat mandible was studied in conjunction with ruthenium red staining. Special care was given to the preservation of proteoglycans in the extracellular matrix. Ruthenium red-positive granules were observed in the pericellular matrix of condylar chondrocytes, and their size and number increased around the hypertrophic cells. However, these granules disappeared in the lowest hypertrophic zone, in which uncalcified cartilage matrix was also disintegrated prior to initiation of ossification. Moreover, hypertrophic chondrocytes observed at the lowest zone appeared intact in their ultrastructural features, i.e., containing numbers of lysosomes and coated vesicles in the cytoplasm facing the blood capillaries. The results strongly suggest that the lowest hypertrophic chondrocytes in rat condylar cartilage may have an active role in the degradation and resorption of the pericellular matrix, especially proteoglycans, and uncalcified matrix, which changes seem an essential step for the initiation of endochondral ossification.     

<Emphasis Type="Italic">Pichia anomala</Emphasis> in grain biopreservation

Cereal grain is a major component of food and feed in large parts of the world. The microbial flora on cereal grains may interfere with hygiene and storage stability, palatability and bioavailability of minerals and proteins may depend on the composition of the microbial population. Therefore, it is of primary interest to control the microbial species present on cereal grain. Inoculation of the biocontrol yeast Pichia anomala to cereal feed grain improved feed hygiene by reduction of moulds and Enterobacteriaceae, and enhanced the nutritional value by increasing the protein content and reducing the concentration of the antinutritional compound phytate. P. anomala strains showed a high phytase activity, for some strains also considerable extracellular phytase activity was observed. A certain maximum in biomass concentration was never exceeded indicating cell density induced growth inhibition of P. anomala.     

Immunohistochemical localization of membrane type 1-matrix metalloproteinase (MT1-MMP) in osteoclasts in vivo

Membrane type 1-matrix metalloproteinase (MT1-MMP) is capable of mediating proteolysis of extracellular matrix. The enzyme has been demonstrated in osteoclasts, in vitro. However, the precise localization in vivo, and therefore the function of the enzyme in osteoclasts, is still unclear. In this study, we immunohistochemically examined the localization of MT1-MMP in rat osteoclasts to clarify the role of MT1-MMP in osteoclastic bone resorption and bone turnover. The localization of MT1-MMP was visualized by the pre-embedding method using anti-MT1-MMP antibody and horseradish peroxidase (HRP) or gold-conjugated antibody. Immunoreactivity of anti-MT1-MMP was found in osteoclasts at the osteoclast-bone interface, but it was not uniform. Ultrastructurally, the immunoreactivity visualized by HRP was found in sealing zone. The plasma membrane at this site showed an irregular border and some invaginations. Immunoreactivity was also found on the surface of certain small vesicles in the cytoplasm. Enhanced silver granules were mainly associated with the sealing membrane. In this study, we demonstrated, for the first time, the localization of MT1-MMP in the sealing zone of osteoclast in vivo. Its distribution suggests that the enzyme modifies the bone surface to facilitate the migration and attachment of osteoclasts as well as scavenging the resorption lacunae.     

播期和种植密度对强、中筋冬小麦蛋白质组分及品质性状的影响

采取裂裂区试验设计,研究了播期和种植密度对强筋小麦临优145和中筋小麦临优2018蛋白质组分和品质性状的影响.结果表明：适期播种的小麦籽粒蛋白质含量和蛋白质产量均最高;推迟播期,强筋品种的醇溶蛋白和谷蛋白含量明显增加,而中筋品种变化不明显;强筋品种的品质性状受播期影响程度高于中筋品种.适期播种,小麦籽粒蛋白质、麦谷蛋白与湿面筋、沉降值、稳定时间、软化度和评价值呈显著或极显著正相关;推迟播期,醇溶蛋白与湿面筋含量呈显著正相关.播期变化引起的蛋白质各组分所占比例的改变是改善小麦品质性状的重要原因.在试验种植密度范围内（225万株·hm-2、300万株·hm^-2和375万株·hm^-2）,小麦籽粒蛋白质含量变化不明显,密度对强筋品种的品质性状有一定调节作用;在低密度条件下（225万株·hm^-2）中筋品种的品质性状最佳.     

Immunohistochemical localization of cathepsins B,D and L in the rat osteoclast

Immunohistochemical localization of cathepsins B, D and L in the osteoclasts of rat alveolar and femoral bones was investigated by using the avidin-biotin-peroxidase complex method for semithin, 1-m-thick cryosections. Extracellular immunoreactivity for cathepsins B and L was clearly demonstrated along the bone resorption lacunae; the intensity of the extracellular immunoreactivity of cathepsin L was stronger than that of cathepsin B. However, the intracellular immunoreactivity of both cathepsins was weak compared with that of cathepsin D. The intracellular immunoreactivity of cathespin D in the osteoclasts was clearly observed in the granules and/or vacuoles, but extracellular cathepsin D immunoreactivity was either negligible or not detected along the resorption lacunae. In the adjacent sections stained with anti-cathepsin L or D, extensive extracellular deposition of cathepsin L was found along the bone resorption lacunae, with or without osteoclasts, although the intracellular reactivity of cathepsin L was weak. This is the first morphological study in which cathepsins B and L have been demonstrated to be produced in the osteoclasts and extensively secreted into resorption lacunae, and in which cathepsin D was found to be present in the cells but scantily secreted into the lacunae. These findings suggest that cathepsins B and L directly and effectively participate in the degradation of the bone matrix.     

Fibroblast activation protein alpha is expressed by chondrocytes following a pro-inflammatory stimulus and is elevated in osteoarthritis

Arthritis is characterised by the proteolytic degradation of articular cartilage leading to a loss of joint function. Articular cartilage is composed of an extracellular matrix of proteoglycans and collagens. We have previously shown that serine proteinases are involved in the activation cascades leading to cartilage collagen degradation. The aim of this study was to use an active-site probe, biotinylated fluorophosphonate, to identify active serine proteinases present on the chondrocyte membrane after stimulation with the pro-inflammatory cytokines IL-1 and oncostatin M (OSM), agents that promote cartilage resorption. Fibroblast activation protein alpha (FAPα), a type II integral membrane serine proteinase, was identified on chondrocyte membranes stimulated with IL-1 and OSM. Real-time PCR analysis shows that FAPα gene expression is up-regulated by this cytokine combination in both isolated chondrocytes and cartilage explant cultures and is significantly higher in cartilage from OA patients compared to phenotypically normal articular cartilage. Immunohistochemistry analysis shows FAPα expression on chondrocytes in the superficial zone of OA cartilage tissues. This is the first report demonstrating the expression of active FAPα on the chondrocyte membrane and elevated levels in cartilage from OA patients. Its cell surface location and expression profile suggest that it may have an important pathological role in the cartilage turnover prevalent in arthritic diseases.     

CARTILAGE RESORPTION IN THE TIBIAL EPIPHYSEAL PLATE OF GROWING RATS

An electron microscopic study of the tibial epiphyseal plates of growing rats reveals that the resorption of unmineralized and mineralized cartilage occurs by two different mechanisms. During resorption the unmineralized transverse cartilaginous walls between chondrocytes are invaded by capillary sprouts. At the resorption zone, numerous cytoplasmic processes derived primarily from the perivascular cells and, to a lesser extent, from the endothelial cells of the sprouts penetrate and appear to lyse the unmineralized transverse cartilaginous walls. Hydrolases released from the degenerating chondrocytes and/or capillary sprouts may also participate in this process. The second resorption mechanism involves the mineralized longitudinal cartilaginous septa. Resorption of these septa is mediated by chondroclasts whose fine structure is identical with that of osteoclasts. The active surface of the chondroclasts has a ruffled border. The surface membrane of the chondroclasts is relatively smooth on either side of the ruffled border and lies in direct apposition with the underlying mineralized cartilage. This observation suggests that the microenvironment in the zone of resorption may be maintained by the neighboring unruffled surfaces of the chondroclasts, which thus seal off and segregate the active portions of these cells.     

Association of leupaxin with Src in osteoclasts

Leupaxin (LPXN), which belongs to the paxillin extended family of adaptor proteins, was previously identified as a component of the sealing zone in osteoclasts. LPXN was found to associate with several podosomal proteins, such as the protein tyrosine kinase Pyk2, the protein-tyrosine phosphatase-PEST (PTP-PEST), actin-binding proteins, and regulators of actin cytoskeletal reorganization. It was previously demonstrated that inhibition of LPXN expression resulted in reduced osteoclast-mediated resorption. In the current study, overexpression of LPXN in murine osteoclasts resulted in both enhanced resorptive activity and cell adhesion, as assessed by in vitro resorption assays. The overexpression of LPXN resulted in an increased association of Pyk2 with LPXN. In an attempt to determine an additional biochemical basis for the observed phenomenon in increased osteoclast activity, a coimmunoprecipitation screen for additional binding partners revealed that Src, a protein tyrosine kinase that is critical to both podosome formation and osteoclast function, was also associated with LPXN. After exposure to the pro-inflammatory and osteoclastogenic cytokine TNF-, there was an increase in the level of Src that coimmunoprecipitated with LPXN. Our data indicate that association of the scaffold protein LPXN with Src adds further complexity to the organization of the podosomal signaling complex in osteoclasts. LPXN; sealing zone; podosomes; LD2 domain