Enzymatic isolation of cells from bone: cytotoxic enzymes of bacterial collagenase

Hefley, Thomas J.; Cushing, Janet E.; Brand, John S.

doi:10.1152/ajpcell.1981.240.5.c234

Cited by 67 publications

(26 citation statements)

References 9 publications

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“…The two polypeptide chains (termed light and heavy) of the native enzyme are encoded by a single 1,581-bp gene, and the junction between the two polypeptide DNA sequences encodes a linker nonapeptide (7). Alpha-clostripain has been implicated in damage of cells in fetal rat calvaria (10) and may contribute to the virulence of other clostridial infections (23). Detection of alpha-clostripain protein and the enzymatic activity in the supernatants of C. perfringens cultures (27) suggest that alpha-clostripain may be a virulence factor.…”

Section: Discussionmentioning

confidence: 99%

Characterization of Genes Regulated Directly by the VirR/VirS System in Clostridium perfringens

et al. 2008

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Analysis of the complete sequence of the genome of Clostridium perfringens strain 13 resulted in identification of five genes, including pfoA (encoding theta toxin) and vrr (encoding VirR/VirS-regulated RNA), with consensus VirR-binding sequences upstream of the open reading frame (ORF), suggesting that expression of these genes may be regulated directly by the two-component VirR/VirS system. To test this possibility, we examined VirR/VirS system-mediated transcriptional regulation of three genes, virT, ccp (encoding alpha-clostripain), and virU, with the novel VirR-binding sequences. Northern analysis revealed that the steady-state levels (increases or decreases in the amounts of RNA expressed) of virT, ccp, and virU mRNAs were lower in a virR mutant strain than in the wild-type strain, as were the levels of the pfoA and vrr transcripts. The consensus VirR-binding sites were located similarly relative to the transcription start sites in the virT, ccp, and virU promoters. Mutation and overexpression analyses with virT and virU revealed that the virT gene product has a negative effect on expression of pfoA and ccp, whereas the virU gene product positively affects expression of pfoA, virT, ccp, and vrr. Nonsense and frameshift mutations in the virT or virU putative ORF did not affect the regulatory functions, suggesting that virT and virU may encode RNA regulators rather than proteins. These results suggest that a complex regulatory network, perhaps involving several regulatory RNA molecules, governs the expression of the VirR/VirS regulon in C. perfringens.

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Section: Discussionmentioning

confidence: 99%

Characterization of Genes Regulated Directly by the VirR/VirS System in Clostridium perfringens

et al. 2008

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“…Osteoblast-enriched Cell Cultures-The parietal bones of 3-5-day-old control and Notch2 Q2319X mutant mice were exposed to type II collagenase from Clostridium histolyticum (Worthington) pretreated with N-␣-tosyl-L-lysyl-chloromethyl ketone hydrochloride at 17 g/ml (Calbiochem) (41). Bones were digested for 20 min at 37°C; cells were extracted in five consecutive reactions, and cells from the last three digestions were pooled and seeded at a density of 10,000 cells/cm 2 , as described (42).…”

Section: Methodsmentioning

confidence: 99%

Hajdu Cheney Mouse Mutants Exhibit Osteopenia, Increased Osteoclastogenesis, and Bone Resorption

Canalis¹,

Schilling²,

Yee

et al. 2016

Journal of Biological Chemistry

106

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Notch receptors are determinants of cell fate and function and play a central role in skeletal development and bone remodeling. Hajdu Cheney syndrome, a disease characterized by osteoporosis and fractures, is associated with NOTCH2 mutations resulting in a truncated stable protein and gain-of-function. We created a mouse model reproducing the Hajdu Cheney syndrome by introducing a 6955C3 T mutation in the Notch2 locus leading to a Q2319X change at the amino acid level. Notch2Q2319X heterozygous mutants were smaller and had shorter femurs than controls; and at 1 month of age they exhibited cancellous and cortical bone osteopenia. As the mice matured, cancellous bone volume was restored partially in male but not female mice, whereas cortical osteopenia persisted in both sexes. Cancellous bone histomorphometry revealed an increased number of osteoclasts and bone resorption, without a decrease in osteoblast number or bone formation. Osteoblast differentiation and function were not affected in Notch2 Q2319X cells. The pre-osteoclast cell pool, osteoclast differentiation, and bone resorption in response to receptor activator of nuclear factor B ligand in vitro were increased in Notch2 Q2319X mutants. These effects were suppressed by the ␥-secretase inhibitor LY450139. In conclusion, Notch2 Q2319X mice exhibit cancellous and cortical bone osteopenia, enhanced osteoclastogenesis, and increased bone resorption.Notch proteins are four single-pass transmembrane receptors that play a critical role in cell fate decisions ( Fig. 1) (1-4). Notch regulates cell renewal and plays a role in skeletal development and homeostasis and in osteoblast and osteoclast differentiation (4 -8). Jagged1 and -2 and DeltaLike1, -3, and -4 are the five classic Notch ligands (4). Notch-ligand interactions result in the proteolytic cleavage and release of the Notch intracellular domain (NICD), 2 which translocates to the nucleus to form a complex with recombination signal-binding protein for immunoglobulin J region (Rbpj) and Mastermind-like to regulate transcription (9 -12). This canonical signaling pathway leads to the transcription of Hairy Enhancer of Split (Hes)1, -5, and -7 and Hes related with YRPW motif (Hey)1, -2, and -L. Skeletal cells express Notch1, Notch2, and low levels of Notch3 transcripts (13-15). Activation of Notch in undifferentiated and differentiated osteoblasts inhibits cell differentiation and function and causes osteopenia (16,17). In contrast, activation of Notch1 in osteocytes causes a pronounced increase in bone mass due to a suppression of bone resorption (18). Results from the conditional inactivation of Notch1 and Notch2 in the developing skeleton confirmed the inhibitory role of Notch in osteoblastogenesis (6,19). Whereas substantial work has characterized the consequences of Notch1 gain-of-function in the skeleton, there is limited knowledge on the function of Notch2 in the postnatal skeleton. This knowledge is particularly important because Notch1 and Notch2 do not have redundant functions, and Notch1 inhibits, wh...

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“…The bones were then digested in 5 ml of the following medium [13]: 25 mM Hepes, 10 mM NdHCO3, 100 mM NaCI, 30 mM KC1,3 mM K 2 H P 0 4 . 3H20, 1 mM CaC12.…”

Section: Isolation Of Primary Bone Cellsmentioning

confidence: 99%

“…2H20, 1 mg/ml bovine serum albumin, 5 mg/ml glucose, 1 mM EDTA, pH 7.4. To this medium was added collagenase at 2.5 mg/ml and 50 pg of a-tosyl-lysyl chloromethane in order to inhibit cytotoxic enzymes present in bacterial collagenase [13]. The bones were digested for 2 h at 37°C in a shaker/water bath after which the medium was separated from the remaining undigested calvariae.…”

Section: Isolation Of Primary Bone Cellsmentioning

confidence: 99%

“…Primary bone cclls obtained from the collagenase digestion of mouse cranial bone [13] were used for immunization and selection procedures to identify monoclonal anti-bodies produced by interspecies hybridization of rat spleen cells with the mouse plasmacytoma line P3X63-Ag8.653 [14]. Antibodies were screened for their ability to inhibit CAMP accumulation in intact mouse cranial bone which had been treated with the 1 -34 amino-terminal peptide of bovine PTH, bPTH .…”

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confidence: 99%

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Identification of a 150‐kDa membrane component which is modulated by parathyroid hormone

Weinshank

Luben

1985

European Journal of Biochemistry

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Monoclonal antibodies have been produced against primary bone cells obtained from the collagenase digestion of mouse cranial bone. Antibodies were selected on the basis of their immunoglobulin class and those which were identified as IgC were further screened for their ability to inhibit CAMP accumulation in response to sub-maximal doses of the 1 -34 amino-terminal peptide of bovine parathyroid hormone, bPTH(1-34). Nine hybridoma clones were subsequently characterized as inhibitory with respect to parathyroid hormone (PTH) responses in intact mouse cranial bone and which also identified a variety of membrane components from detergent extracts of surface-labeled primary bone cells. Five of these antibodies immunoprecipitated a membrane component with M , of 80000 that appeared to be a major component of the extract susceptible to surface-labeling with lZ51. All nine monoclonal antibodies were shown to bind to a suspcndcd-cell preparation of primary bone cells with 2 -3 orders of magnitude greater binding than that of control antibodies. Using this assay, one clone, designated 3G12 IgG, was observed to exhibit desensitization effects at the binding level with a time course and dose dependency for PTH pre-incubation that was similar to the establishment of the refractory state in other systems. In addition, the desensitization effect oCcurred at 37°C but not at 4°C. This antibody was shown to bind saturably to both intact mouse cranial bone and primary bone cells with an apparent affinity constant (Ka) in the range of lo9 M. Inhibition of bone CAMP accumulation in response to 2.5 nM bPTH(1-34) was directly correlated to the binding of 3G12 IgC to intact mouse calvariae. A niaximuiii inhibition of approximately 85% was obscrved. 3G12 IgC iinmunoprecipitated a single membrane component, M , 150000, from NP-40 detergent extracts of "sI-labeled primary mouse bone cells. The molecular mass of this component was also 150000 daltons when run on polyacrylamide gel slabs under non-reducing conditions. Control and PTH-pre-treated bone cells were surfacelabeled, detergent-solubilized and immunoprecipitated with 3G12 IgG in order to investigate further the desensitization effect at the molecular level. Incubation of bone cells with 1 pg/ml bPTH(1 -34) for 45 min at 37 C caused an increased susceptibility to surface-labeling with IZ5I that was approximately three-fold higher in specific activity than that of control cells. Under conditions of quantitative iminunoprccipitation, 3GI 2 IgG precipitated less of the 150-kDa membrane component from the PTH pre-treated bone cell extract, indicating a decreased availability of this membrane component under conditions of desensitization. We propose that the 150-kDa protein from mouse bone cells may be the PTH receptor, a component of the receptor or a membrane protein which is directly involved in the PTH responsiveness of these cells.Parathyroid hormone (PTH) is a peptide hormone which initiates a variety of biological activities by binding to specific cell-surface receptors prcscnt on...

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Enzymatic isolation of cells from bone: cytotoxic enzymes of bacterial collagenase

Cited by 67 publications

References 9 publications

Characterization of Genes Regulated Directly by the VirR/VirS System in Clostridium perfringens

Characterization of Genes Regulated Directly by the VirR/VirS System in Clostridium perfringens

Hajdu Cheney Mouse Mutants Exhibit Osteopenia, Increased Osteoclastogenesis, and Bone Resorption

Identification of a 150‐kDa membrane component which is modulated by parathyroid hormone

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