IN VIVO SERIAL CHANGES IN BONE INDUCTION BY E. COLI-DERIVED RECOMBINANT HUMAN BMP-2

2002 ◽  
Vol 06 (01) ◽  
pp. 1-7 ◽  
Author(s):  
Kazuhisa Bessho ◽  
Junya Sinobe ◽  
Shinji Kaihara ◽  
Mariko Kawai ◽  
Yasunori Okubo ◽  
...  
Keyword(s):  
High Activity ◽  
Mammalian Cells ◽  
Type I Collagen ◽  
Expression System ◽  
Chinese Hamster ◽  
Bacterial Expression ◽  
Ovary Cell ◽  
Type I ◽  
Bacterial Expression System

At present, several recombinant human bone morphogenetic proteins (rhBMPs) can be produced in mammalian cells. If rhBMPs with a high activity could be produced in bacteria, the bacterial expression system is very useful in clinic. We examined in vivo serial changes in the bone inducing ability of an Escherichia coli-derived rhBMP-2 (ErhBMP-2) variant with N-terminal sequence. Five μg of ErhBMP-2 was mixed with 3 mg of atelopeptide type I collagen (CL) as the carrier, and specimens were implanted into calf muscle pouches of Wistar rats (n = 20). After 3, 7, 14 and 21 days, 5 specimens each were examined. New cartilage was observed 7 days after implantation of ErhBMP-2 with CL. Induced bone was found on the outermost edge of the implant after 14 days. After 21 days, bone formation was associated with much fatty marrow. ALP activity and calcium content gradually increased with time. These changes were similar to those in a study using Chinese hamster ovary cell-derived rhBMP-2. However, these increases were slightly higher than those in the rhBMP-2 study. From these findings, ErhBMP-2 appears to be completely renatured while maintaining its biological activity. ErhBMP-2 with CL may expand by absorbing body fluid in vivo to greater extent than rhBMP-2 with CL, because it lacks heparin-binding sites. A variant ErhBMP-2 showing high activity in vivo, obtained by the bacterial expression system is an important finding, as it should be possible to produce large quantities of ErhBMP-2 at a low-cost for clinical use, in the near future.

Modulative Effects of Metabolic Effectors on Benzo(A)Pyrene-Induced Cytotoxicity and Mutagenicity in Mammalian Cells

1994 ◽  
Vol 10 (3) ◽  
pp. 181-189 ◽  
Author(s):  
Abraham W. Hsie ◽  
Leslie Recio
Keyword(s):  
Mammalian Cells ◽  
Glucuronic Acid ◽  
Chinese Hamster ◽  
Ovary Cell ◽  
Future Research ◽  
Uridine Diphosphate ◽  
Glucuronide Conjugation ◽  
Hypoxanthine Guanine Phosphoribosyltransferase ◽  
Mutagenic Effects

Conjugation and detoxification of mixed function oxidase (MFO)-mediated benzo(a)pyrene [B(a)P] metabolites with glucuronic acid and glutathione (GSH) are major pathways of B(a)P elimination and ultimately excretion in vivo. We have studied the effects of uridine diphosphate α-D-glucuronic acid (UDPGA) and GSH, a cofactor for the synthesis of glucuronide and GSH conjugates, respectively, on B(a)P-induced cytotoxicity and mutagenicity in mammalian cells. The S9-mix used in the Chinese hamster ovary cell/hypoxanthine-guanine phosphoribosyltransferase (CHO/HPRT) mutational assay was supplemented with either UDPGA, GSH, or GSH plus purified GSH-S-transferases (GSHTs), to study modulation of glucuronide and GSH detoxification mechanisms on B(a)P-induced cytotoxic and mutagenic effects. We found that the addition of UDPGA to S9-mix reduces cytotoxicity induced by either B(a)P or B(a)P 6-OH but not by B(a)P 7,8-diol [B(a)P-diol]. The reduction of B(a)P and B(a)P 6-OH-induced cytotoxicity by glucuronide conjugation is likely due to elimination of cytotoxic phenols and quinones. The addition of GSH to the S9-mix resulted in a reduction of B(a)P- and B(a)P-diol-induced cytotoxicity. GSH plus GSHT reduced B(a)P-induced cytotoxicity and mutagenicity. GSH inhibited the mutagenicity at low concentrations of B(a)P-diol. GSH plus GSHTs inhibited the cytotoxicity and mutagenicity of B(a)P-diol at concentrations not affected by GSH alone. These studies demonstrate that mechanisms of detoxification can affect the biological activity of B(a)P and B(a)P-diol as profoundly as bioactivation by the MFO system. Future research should address studies of mutagenicity modulation by metabolic effectors at both the molecular (DNA sequence) and cellular (quantitative mutagenesis) level.

scholarly journals Activation of a mammalian origin of replication by chromosomal rearrangement.

1992 ◽  
Vol 12 (6) ◽  
pp. 2804-2812 ◽  
Author(s):  
T H Leu ◽  
J L Hamlin
Keyword(s):  
Dihydrofolate Reductase ◽  
Mammalian Cells ◽  
Chromosomal Rearrangement ◽  
Chinese Hamster ◽  
Regulatory Elements ◽  
Replication Initiation ◽  
Type I ◽  
Type Ii ◽  
Direct Demonstration

The methotrexate-resistant Chinese hamster cell line DC3F/A3-4K (A3/4K) contains at least two prominent dihydrofolate reductase amplicon types. The type I amplicons, constituting approximately 80% of the total, are at least 650 kb in length, but the endpoints have not yet been characterized. The type II sequences represent approximately 20% of amplicons, are 450 kb in length, and are arranged as alternating head-to-head and tail-to-tail repeats. In previous studies on the CHOC 400 line, in which the amplicons are much smaller, a replication initiation locus (ori-beta/ori-gamma) has been shown to reside downstream from the dihydrofolate reductase gene. In a more recent study on the larger amplicons of A3/4K cells, we detected an additional initiation locus (ori-alpha) lying approximately 240 kb upstream from ori-beta/ori-gamma. Interestingly, in vivo labelling experiments suggested that replication forks diverge from ori-alpha only in the downstream direction. This finding suggested either that ori-alpha is a unidirectional origin or that a terminus lies immediately upstream from ori-alpha. However, in this study, we show that ori-alpha is actually very close to the head-to-head palindromic junction sequence between the minor type II amplicons in A3/4K cells; furthermore, ori-alpha is active in the early S period in the type II amplicons but not in the larger type I sequences that lack this palindromic junction. This is the first direct demonstration in mammalian cells that a cryptic origin can be activated by chromosomal rearrangement, presumably by deleting negative regulatory elements or by creating a more favorable chromosomal milieu for initiation.

scholarly journals Activation of a mammalian origin of replication by chromosomal rearrangement

1992 ◽  
Vol 12 (6) ◽  
pp. 2804-2812
Author(s):  
T H Leu ◽  
J L Hamlin
Keyword(s):  
Dihydrofolate Reductase ◽  
Mammalian Cells ◽  
Chromosomal Rearrangement ◽  
Chinese Hamster ◽  
Regulatory Elements ◽  
Replication Initiation ◽  
Type I ◽  
Type Ii ◽  
Direct Demonstration

The methotrexate-resistant Chinese hamster cell line DC3F/A3-4K (A3/4K) contains at least two prominent dihydrofolate reductase amplicon types. The type I amplicons, constituting approximately 80% of the total, are at least 650 kb in length, but the endpoints have not yet been characterized. The type II sequences represent approximately 20% of amplicons, are 450 kb in length, and are arranged as alternating head-to-head and tail-to-tail repeats. In previous studies on the CHOC 400 line, in which the amplicons are much smaller, a replication initiation locus (ori-beta/ori-gamma) has been shown to reside downstream from the dihydrofolate reductase gene. In a more recent study on the larger amplicons of A3/4K cells, we detected an additional initiation locus (ori-alpha) lying approximately 240 kb upstream from ori-beta/ori-gamma. Interestingly, in vivo labelling experiments suggested that replication forks diverge from ori-alpha only in the downstream direction. This finding suggested either that ori-alpha is a unidirectional origin or that a terminus lies immediately upstream from ori-alpha. However, in this study, we show that ori-alpha is actually very close to the head-to-head palindromic junction sequence between the minor type II amplicons in A3/4K cells; furthermore, ori-alpha is active in the early S period in the type II amplicons but not in the larger type I sequences that lack this palindromic junction. This is the first direct demonstration in mammalian cells that a cryptic origin can be activated by chromosomal rearrangement, presumably by deleting negative regulatory elements or by creating a more favorable chromosomal milieu for initiation.

Fibulin-1 suppression of fibronectin-regulated cell adhesion and motility

2001 ◽  
Vol 114 (24) ◽  
pp. 4587-4598 ◽  
Author(s):  
Waleed O. Twal ◽  
Andras Czirok ◽  
Balazs Hegedus ◽  
Christian Knaak ◽  
Mastan R. Chintalapudi ◽  
...  
Keyword(s):  
Matrix Protein ◽  
Type I Collagen ◽  
Cell Attachment ◽  
Chinese Hamster ◽  
Extracellular Matrix Protein ◽  
Endocardial Cushion ◽  
Boyden Chamber ◽  
Type I ◽  
Collagen Gels

Fibulin-1 is an extracellular matrix protein often associated with fibronectin (FN) in vivo. In this study, the ability of fibulin-1 to modulate adhesion, spreading and motility-promoting activities of FN was investigated. Fibulin-1 was found to have pronounced inhibitory effects on the cell attachment and spreading promoted by FN. Fibulin-1 was also found to inhibit the motility of a variety of cell types on FN substrata. For example, the FN-dependent haptotactic motility of breast carcinoma (MDA MB231) cells, epidermal carcinoma (A431), melanoma (A375 SM), rat pulmonary aortic smooth muscle cells (PAC1) and Chinese hamster ovary (CHO) cells was inhibited by the presence of fibulin-1 bound to FN-coated Boyden chamber membranes. Cells transfected to overproduce fibulin-1 displayed reduced velocity, distance of movement and persistence time on FN substrata. Similarly, the incorporation of fibulin-1 into FN-containing type I collagen gels inhibited the invasion of endocardial cushion mesenchymal cells migrating from cultured embryonic heart explants. By contrast, incorporation of fibulin-1 into collagen gels lacking FN had no effect on the migration of endocardial cushion cells. These results suggest that the motility-suppressive effects of fibulin-1 might be FN specific. Furthermore, such effects are cell-type specific, in that the migration of gingival fibroblasts and endothelial cells on FN substrata is not responsive to fibulin-1. Additional studies found that the mechanism for the motility-suppressive effects of fibulin-1 does not involve perturbations of interactions between α5β1 or α4 integrins, or heparan sulfate proteoglycans with FN. However, fibulin-1 was found to inhibit extracellular signal regulated kinase (ERK) activation and to suppress phosphorylation of myosin heavy chain. This ability to influence signal transduction cascades that modulate the actin-myosin motor complex might be the basis for the effects of fibulin-1 on adhesion and motility.

scholarly journals Effects of chitosan-collagen dressing on wound healing in vitro and in vivo assays

2021 ◽  
Vol 19 ◽  
pp. 228080002198969
Author(s):  
Min-Xia Zhang ◽  
Wan-Yi Zhao ◽  
Qing-Qing Fang ◽  
Xiao-Feng Wang ◽  
Chun-Ye Chen ◽  
...  
Keyword(s):  
Wound Healing ◽  
Wound Dressing ◽  
Type I Collagen ◽  
Inhibition Zone ◽  
Negative Control ◽  
Type I ◽  
Nih3t3 Cells ◽  
Post Operation

The present study was designed to fabricate a new chitosan-collagen sponge (CCS) for potential wound dressing applications. CCS was fabricated by a 3.0% chitosan mixture with a 1.0% type I collagen (7:3(w/w)) through freeze-drying. Then the dressing was prepared to evaluate its properties through a series of tests. The new-made dressing demonstrated its safety toward NIH3T3 cells. Furthermore, the CCS showed the significant surround inhibition zone than empty controls inoculated by E. coli and S. aureus. Moreover, the moisture rates of CCS were increased more rapidly than the collagen and blank sponge groups. The results revealed that the CCS had the characteristics of nontoxicity, biocompatibility, good antibacterial activity, and water retention. We used a full-thickness excisional wound healing model to evaluate the in vivo efficacy of the new dressing. The results showed remarkable healing at 14th day post-operation compared with injuries treated with collagen only as a negative control in addition to chitosan only. Our results suggest that the chitosan-collagen wound dressing were identified as a new promising candidate for further wound application.

scholarly journals Neurotrophin Receptor TrkC Is an Entry Receptor for Trypanosoma cruzi in Neural, Glial, and Epithelial Cells

2011 ◽  
Vol 79 (10) ◽  
pp. 4081-4087 ◽  
Author(s):  
Craig Weinkauf ◽  
Ryan Salvador ◽  
Mercio PereiraPerrin
Keyword(s):  
Trypanosoma Cruzi ◽  
Chagas Disease ◽  
Mammalian Cells ◽  
Chinese Hamster ◽  
Neuronal Cell ◽  
Host Cells ◽  
Neurotrophin Receptor ◽  
Content Type

ABSTRACTTrypanosoma cruzi, the agent of Chagas' disease, infects a variety of mammalian cells in a process that includes multiple cycles of intracellular division and differentiation starting with host receptor recognition by a parasite ligand(s). Earlier work in our laboratory showed that the neurotrophin-3 (NT-3) receptor TrkC is activated byT. cruzisurfacetrans-sialidase, also known as parasite-derived neurotrophic factor (PDNF). However, it has remained unclear whether TrkC is used byT. cruzito enter host cells. Here, we show that a neuronal cell line (PC12-NNR5) relatively resistant toT. cruzibecame highly susceptible to infection when overexpressing human TrkC but not human TrkB. Furthermore,trkCtransfection conferred an ∼3.0-fold intracellular growth advantage. Sialylation-deficient Chinese hamster ovarian (CHO) epithelial cell lines Lec1 and Lec2 also became much more permissive toT. cruziafter transfection with thetrkCgene. Additionally, NT-3 specifically blockedT. cruziinfection of the TrkC-NNR5 transfectants and of naturally permissive TrkC-bearing Schwann cells and astrocytes, as did recombinant PDNF. Two specific inhibitors of Trk autophosphorylation (K252a and AG879) and inhibitors of Trk-induced MAPK/Erk (U0126) and Akt kinase (LY294002) signaling, but not an inhibitor of insulin-like growth factor 1 receptor, abrogated TrkC-mediated cell invasion. Antibody to TrkC blockedT. cruziinfection of the TrkC-NNR5 transfectants and of cells that naturally express TrkC. The TrkC antibody also significantly and specifically reduced cutaneous infection in a mouse model of acute Chagas' disease. TrkC is ubiquitously expressed in the peripheral and central nervous systems, and in nonneural cells infected byT. cruzi, including cardiac and gastrointestinal muscle cells. Thus, TrkC is implicated as a functional PDNF receptor in cell entry, independently of sialic acid recognition, mediating broadT. cruziinfection bothin vitroandin vivo.

Vitamin K promotes mineralization, osteoblast-to-osteocyte transition, and an anticatabolic phenotype by γ-carboxylation-dependent and -independent mechanisms

2009 ◽  
Vol 297 (6) ◽  
pp. C1358-C1367 ◽  
Author(s):  
Gerald J. Atkins ◽  
Katie J. Welldon ◽  
Asiri R. Wijenayaka ◽  
Lynda F. Bonewald ◽  
David M. Findlay
Keyword(s):  
Bone Formation ◽  
Vitamin K ◽  
Type I Collagen ◽  
Vitamin K2 ◽  
Type I ◽  
Vitamin K1 ◽  
Vitamin K3 ◽  
Positive Effect

The vitamin K family members phylloquinone (vitamin K1) and the menaquinones (vitamin K2) are under study for their roles in bone metabolism and as potential therapeutic agents for skeletal diseases. We have investigated the effects of two naturally occurring homologs, phytonadione (vitamin K1) and menatetrenone (vitamin K2), and those of the synthetic vitamin K, menadione (vitamin K3), on human primary osteoblasts. All homologs promoted in vitro mineralization by these cells. Vitamin K1-induced mineralization was highly sensitive to warfarin, whereas that induced by vitamins K2 and K3 was less sensitive, implying that γ-carboxylation and other mechanisms, possibly genomic actions through activation of the steroid xenobiotic receptor, are involved in the effect. The positive effect on mineralization was associated with decreased matrix synthesis, evidenced by a decrease from control in expression of type I collagen mRNA, implying a maturational effect. Incubation in the presence of vitamin K2 or K3 in a three-dimensional type I collagen gel culture system resulted in increased numbers of cells with elongated cytoplasmic processes resembling osteocytes. This effect was not warfarin sensitive. Addition of calcein to vitamin K-treated cells revealed vitamin K-dependent deposition of mineral associated with cell processes. These effects are consistent with vitamin K promoting the osteoblast-to-osteocyte transition in humans. To test whether vitamin K may also act on mature osteocytes, we tested the effects of vitamin K on MLO-Y4 cells. Vitamin K reduced receptor activator of NF-κB ligand expression relative to osteoprotegerin by MLO-Y4 cells, an effect also seen in human cultures. Together, our findings suggest that vitamin K promotes the osteoblast-to-osteocyte transition, at the same time decreasing the osteoclastogenic potential of these cells. These may be mechanisms by which vitamin K optimizes bone formation and integrity in vivo and may help explain the net positive effect of vitamin K on bone formation.

Cells in regenerating deer antler cartilage provide a microenvironment that supports osteoclast differentiation

2001 ◽  
Vol 204 (3) ◽  
pp. 443-455
Author(s):  
C. Faucheux ◽  
S. Nesbitt ◽  
M. Horton ◽  
J. Price
Keyword(s):  
Type I Collagen ◽  
Mononuclear Cells ◽  
Osteoclast Differentiation ◽  
Cathepsin K ◽  
Collagen Matrix ◽  
Tartrate Resistant Acid Phosphatase ◽  
Type I ◽  
Deer Antler

Deer antlers are a rare example of mammalian epimorphic regeneration. Each year, the antlers re-grow by a modified endochondral ossification process that involves extensive remodelling of cartilage by osteoclasts. This study identified regenerating antler cartilage as a site of osteoclastogenesis in vivo. An in vitro model was then developed to study antler osteoclast differentiation. Cultured as a high-density micromass, cells from non-mineralised cartilage supported the differentiation of large numbers of osteoclast-like multinucleated cells (MNCs) in the absence of factors normally required for osteoclastogenesis. After 48 h of culture, tartrate-resistant acid phosphatase (TRAP)-positive mononuclear cells (osteoclast precursors) were visible, and by day 14 a large number of TRAP-positive MNCs had formed (783+/−200 per well, mean +/− s.e.m., N=4). Reverse transcriptase/polymerase chain reaction (RT-PCR) showed that receptor activator of NF κ B ligand (RANKL) and macrophage colony stimulating factor (M-CSF) mRNAs were expressed in micromass cultures. Antler MNCs have the phenotype of osteoclasts from mammalian bone; they expressed TRAP, vitronectin and calcitonin receptors and, when cultured on dentine, formed F-actin rings and large resorption pits. When cultured on glass, antler MNCs appeared to digest the matrix of the micromass and endocytose type I collagen. Matrix metalloproteinase-9 (MMP-9) may play a role in the resorption of this non-mineralised matrix since it is highly expressed in 100 % of MNCs. In contrast, cathepsin K, another enzyme expressed in osteoclasts from bone, is only highly expressed in resorbing MNCs cultured on dentine. This study identifies the deer antler as a valuable model that can be used to study the differentiation and function of osteoclasts in adult regenerating mineralised tissues.

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