Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.2.1.23 (beta-galactosidase)
14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

An enzyme immunoassay for the direct measurement of elcatonin, a synthetic analogue of eel calcitonin with hypocalcemic activity, in rat and dog plasma was developed. Elcatonin was conjugated to beta-galactosidase using an N-succinimidyl ester, and the product was used as the tracer. The antibody-bound tracer was precipitated using a second antibody. The nonequilibrated procedure gave better sensitivity and precision than the equilibrated procedure. The nonequilibrated method was sensitive at quantifiable concentrations as low as 0.2 ng/mL using 0.1 mL of plasma and was also reproducible, with RSD values generally less than +/- 10%. Plasma levels in rats and dogs were determined by this competitive enzyme immunoassay after intramuscular or intravenous injection of 40 U/kg (8 micrograms/kg) of elcatonin.
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PMID:Enzyme immunoassay for elcatonin, a synthetic analogue of eel calcitonin, in plasma. 240 65

The relationships both between cholinergic neurons and substance P (SP) and between cholinergic neurons and calcitonin gene-related peptide (CGRP) terminals were examined in the rat sacral intermediolateral nucleus at the light and electron microscopic levels by means of double-immunostaining methods. Cholinergic neurons were labeled by a monoclonal antibody to choline acetyltransferase (CAT) with the avidin-biotin technique and stained bluish-green by indolyl-beta-galactoside reaction products with beta-galactosidase as a marker. On the same sections, SP or CGRP fibers were labeled by polyclonal antisera to SP or CGRP after application of the peroxidase-antiperoxidase (PAP) method and stained brown by the p-dimethylaminoazobenzene (DAB) reaction. After embedding in Epon, light and electron microscopic sections were examined. At the light microscopic level, CGRP-like immunoreactive (CGRP-I) fibers and SP-like immunoreactive (SP-I) fibers were found to pass through the lateral edge of the dorsal horn and then into the dorsal region of the sacral intermediolateral nucleus. In addition, SP-I fibers also extend from the dorsolateral funiculus into the entire sacral intermediolateral region. At the electron microscopic level, many axosomatic and axodendritic synapses were found between CAT-I structures and SP-I terminals in the intermediolateral nucleus, whereas most of the CGRP-I terminals in this area made axodendritic synapses with CAT-I dendrites. These results indicate that cholinergic neurons in the sacral intermediolateral nucleus receive direct synaptic input from SP-I and CGRP-I terminals.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Interaction between cholinergic neurons and substance P or calcitonin gene-related peptide terminals of the rat sacral intermediolateral nucleus: double immunostaining at the light and electron microscopic levels. 247 8

The human breast carcinoma cell line T47D is known to express high-affinity calcitonin receptors (CTRs). PCR amplification of the CTR cDNA from T47D mRNA resulted in the identification of two different cDNAs that encode distinct receptor isoforms, h alpha CTR and h beta CTR. The two cDNAs are identical except that the h alpha CTR cDNA contains a 48 bp insert sequence that encodes a 16 amino acid domain in the first cytosolic loop of the receptor. Stable transfection of each receptor cDNA into murine erythroleukaemia (MEL) cells resulted in the expression of receptors with high affinity for radiolabelled salmon calcitonin (h alpha CTR Kd 0.09 nM, h beta CTR Kd 0.12 nM). Ligand competition binding studies did not reveal any significant pharmacological difference between the receptor isoforms. In transfected MEL cells and COS-1 cells the h beta CTR isoform was expressed at tenfold higher levels than the h alpha CTR. A reporter gene assay that monitored the coupling of CTR to adenylate cyclase by increases in beta-galactosidase activity indicated that both receptors were able to stimulate cyclic AMP production in response to ligand binding.
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PMID:Identification of multiple human calcitonin receptor isoforms: heterologous expression and pharmacological characterization. 761 7

Human calcitonin (hCT) is a C-terminus alpha-amidated peptide hormone consisting of 32 amino acids. The amidated structure is essential for its biological activities, and the C-terminal-glycine-extended precursor peptide, hCT[G], is converted to bioactive hCT by a C-terminus-alpha-amidating enzyme. An efficient production method is described for the hCT[G] peptide, as a part of the fusion protein consisting of a modified E. coli beta-galactosidase, linker amino acids and hCT[G]. Stable inclusion bodies of the fusion protein in E. coli were expressed by focusing on the amino acid charge, and the fusion protein was modified by inserting a basic amino acid sequence into its linker region. This modification greatly affected the formation of inclusion bodies. E. coli strain W3110/pG97S4DhCT [G]R4 could produce a large amount of stable inclusion bodies, and the hCT[G] peptide was released quantitatively from the fusion protein by S. aureus V8 protease. This enabled a large-scale production method to be established for the hCT[G] precursor peptide in E. coli to produce mature hCT.
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PMID:High expression of a recombinant human calcitonin precursor peptide in Escherichia coli. 776 11

Insulin-like growth factor-I (IGF-I) and IGF-II have powerful, well defined effects on osteoblastic cells, stimulating their proliferation and inducing collagen synthesis, but the role of IGF-I and -II in modulating osteoclast differentiation and activity remains unclear. We first examined the bone-resorptive effects of IGF-I and IGF-II by assessing 45Ca2+ release from neonatal mouse calvarial bones. Both IGFs dose dependently stimulated bone resorption, with an EC50 of 8 x 10(-9) M for IGF-I and 2 x 10(-8) M for IGF-II. We then tested the effects of the IGFs on bone resorption by rat isolated osteoclasts cultured on ivory slices. Neither IGF-I nor IGF-II stimulated isolated osteoclast activity. However, in the presence of either primary mouse osteoblasts or human osteosarcoma MG 63 cells, both IGFs enhanced osteoclast resorptive activity, with an EC50 of 5 x 10(-10) M for IGF-I and 10(-9) M for IGF-II. Stimulation was not mediated by BALB/c/3T3 cells, a nonosteoblastic cell line. The effects of the IGFs were blocked by alpha IR-3, an antibody to the type I IGF receptor, but not by beta-galactosidase, a lysosomal enzyme that competes with IGF-II for the type II IGF receptor. We then examined the effects of the IGFs on the formation of osteoclast-like multinucleate cells (MNCs) in mouse bone marrow cultures. IGF-I and -II dose dependently increased the number of tartrate-resistant acid phosphatase (TRAP)-positive MNCs, although their effects were less than that of 1,25-dihydroxyvitamin D3 (a hormone that induces osteoclast differentiation). No TRAP-positive MNCs appeared in the absence of these hormones. Like authentic osteoclasts, the TRAP-positive MNCs formed in response to IGF-I and -II bound [125I]salmon calcitonin. When mouse bone marrow cells were cultured on ivory slices in the presence of either IGF-I or IGF-II for 10 days, numerous resorption lacunae were formed. beta-Galactosidase had no effect on IGF-mediated osteoclast formation. These results are strong evidence that both IGF-I and IGF-II stimulate bone resorption in vitro by enhancing osteoclast formation and function. Our data also suggest that the IGFs act through the intermediary of osteoblastic cells to stimulate osteoclast activity and that the type I, but not the type II, IGF receptor is involved in their responses. We propose that the local production of IGF-I and IGF-II may modulate both osteoblast-osteoclast interactions and osteoclast formation and play an important role in bone remodeling.
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PMID:Osteoblasts mediate insulin-like growth factor-I and -II stimulation of osteoclast formation and function. 782 21

A sandwich transfer enzyme immunoassay for salmon calcitonin (SCT) and its usability for the pharmacokinetic study are described. The assay procedure consisted of the reaction of SCT with 2,4-dinitrophenyl biotinyl anti-SCT IgG and anti-SCT Fab'-beta-galactosidase conjugate, trapping onto (anti-2,4-dinitrophenyl bovine serum albumin) IgG-coated polystyrene balls, eluting with epsilon N-2,4-dinitrophenyl-L-lysine and transferring to streptavidin-coated polystyrene balls and fluorometric detection of beta-D-galactosidase activity. The practical detection limit of SCT was 0.05 pg (15 amol)/50 microliters of sample and 1 pg/ml as the concentration. The application of this method has enabled us to directly estimate the bioavailability of SCT dosed intranasally at the therapeutic level (160 IU, 31 micrograms) for its anti-osteoporotic effect as compared to an intramuscular dose (10 IU, 1.9 micrograms). The pharmacokinetic parameters of the intranasal SCT (n = 6) thus estimated were as follows: the area under the blood concentration-time curve (AUC) 9400 +/- 5400 (SD) pg.h/ml, and the mean residence time (MRT) = 42 +/- 14 (SD) min, when the AUC for the intramuscular SCT (n = 3) = 5600 +/- 2000 (SD) pg.h/ml and the MRT = 39 +/- 19 (SD) min.
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PMID:A sandwich transfer enzyme immunoassay for salmon calcitonin: determination of the bioavailability of intranasal salmon calcitonin in human. 940 61

Progress in the field of osteoclast gene regulation has been hampered significantly by the lack of such cell lines. In this study, mouse osteoclast precursor cells were elicited in an osteoclast-inductive coculture system and immortalized using SV40 large T antigen. One of the osteoclast precursor cell lines (MOCP-5) forms 95% tartrate-resistant acid phosphatase positive (TRAP+) multinuclear osteoclast-like cells (OCLs) in the coculture system. The yield of TRAP+ OCLs was 4.5-7x10(4) cells per 10 cm2 dish. Expression of SV40 large T antigen was visualized in the nucleus of MOCP-5 cells and OCLs by immunohistochemistry. MOCP-5 cells were positive for MoMa-2 antigen and nonspecific esterase but negative for F4/80 antigen. OCLs derived from MOCP-5 cells were positive for able to form extensive resorption bone pits on bone slices. The resorbing activity of the OCLs was comparable to that of authentic mouse osteoclasts. Pit formation was inhibited by salmon calcitonin (CT). Acid production by OCLs was demonstrated by vital staining with acridine orange. The OCLs expressed cathepsin K and CT receptors. MOCP-5 cells could be transfected by a construct that carries the beta-galactosidase gene. Transfected MOCP-5 cells expressing beta-galactosidase retain the ability to differentiate into OCLs, indicating a useful model for osteoclast gene regulation. To date, the MOCP-5 cell line has been maintained in continuous culture for 23 months and has maintained the capacity to differentiate into osteoclasts throughout this time. In summary, these data show that a stable immortalized osteoclast precursor cell line has been established and that the immortalization with SV40 large T oncogene does not prevent osteoclast precursor cell differentiation.
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PMID:Generation of mouse osteoclastogenic cell lines immortalized with SV40 large T antigen. 966 Oct 75

We have investigated the transcriptional regulation of the porcine calcitonin (CT) receptor (pCTR) promoter in transgenic mice. A construct containing 2.1 kb pCTR 5' flanking region, fused to a beta-galactosidase (lacZ) gene, was employed for the production of transgenic mice. At 11.5 days of development lacZ expression was observed in the embryonic brain and spinal cord. By 15.5 days post fertilization, lacZ expression was detected in the developing mammary gland, external ear, cartilage primordium of the humerus, and anterior naris (nostril). RT-PCR on RNA from these fetal tissues showed endogenous mouse CTR (mCTR) expression. In neonatal and adult transgenics, lacZ expression was silenced, except in brain, spinal cord, and testis (adults only). Endogenous mCTR gene expression and pCTR promoter activity were corepressed in the same tissues from adult mice. No pCTR promoter activity was detected in the kidney or bone of transgenic animals. This suggests that additional DNA sequences may be required for pCTR promoter activity in these tissues. From these results, we conclude that the pCTR promoter is active only in tissues expressing endogenous mCTR. Many of the these tissues represent previously unknown sites of CTR gene expression. Finally, the developmental regulation of pCTR/mCTR in tissues such as breast and cartilage primordium suggests that CTRs may play a role in the morphogenesis of these tissues.
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PMID:The porcine calcitonin receptor promoter directs expression of a linked reporter gene in a tissue and developmental specific manner in transgenic mice. 988 62

Herpes simplex virus thymidine kinase (HSVtk) gene transfer followed by ganciclovir administration is a common strategy for experimental cancer therapy. To evaluate the feasibility of using the human calcitonin promoter to target medullary thyroid carcinoma (MTC), we developed adenovirus vectors containing Escherichia coli beta-galactosidase gene under the control of the CALC-I promoter (AdCTlacZ), or the human cytomegalovirus promoter (AdCMVlacZ). Beta-galactosidase activity driven by the CALC-I promoter was higher than by the CMV promoter in rat MTC cells after infection with adenovirus vectors. AdCTlacZ induced an equal or lower expression level of beta-galactosidase in TT (human MTC), T98G, Cos1, HepG2, and HeLa cells compared with AdCMVlacZ. To inhibit the growth of MTC cells, we developed two adenovirus vectors, AdCMVtk carrying HSVtk driven by the cytomegalovirus promoter and AdDCTtk containing a human CALC-I minigene under the control of the CALC-I promoter. HSVtk is fused to a portion of calcitonin coded in exon 4 to direct cell-specific regulation of splicing. All cell lines infected with AdCMVtk were rendered sensitive to ganciclovir, whereas T98G and Cos1 cells infected with AdDCTtk were not affected. Cell killing was also observed in HeLa, HepG2, rat MTC and TT cells infected with AdDCTtk.
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PMID:Cell-specific induction of sensitivity to ganciclovir in medullary thyroid carcinoma cells by adenovirus-mediated gene transfer of herpes simplex virus thymidine kinase. 1080 92

In order to efficiently recover recombinant proteins, a temperature-sensitive lytic system was constructed on the basis of the feature that T4 lysozyme disrupts the bacteria through cutting specific bond in the peptidoglycan layer of cell wall. This system was evaluated by constructing and introducing a low copy plasmid pSC-lys (pSC101 replication origin) into E.coli. The plasmid contained a temperature sensitive T4 lysozyme (LYS(ts)) gene under the control of three tandem tac promoters and the LacI repressor, which is compatible with other plasmids carrying pMB1, ColE1 replication origins, etc. Under the optimum lysis conditions, 2--5 fold condensed cultures resuspended in buffer A, beta-galactosidase, recombinant chaperone GroEL and ZZ-fusion salmon hexamic calcitonin (Cal6) in E.coli were released simply, rapidly, and quantitatively, as co-expressed with LYS(ts). The two tested recombinant proteins maintained their significant productions. Instead of other cumbersome lysising methods, this novel lytic system will be useful in recovery of recombinant proteins for further purification in the field of biotechnology.
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PMID:The Application of a Novel Lytic System to the Recovery of Recombinant Proteins in E.coli. 1207 42


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