Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Human neutrophil collagenase splice variant

a neutrophil and collagenase technology, applied in the field of human neutrophil collagenase splice variants, to achieve the effect of preventing the expression of human mmp-8alt and reducing or preventing the effect of human mmp-8alt polypeptides

Inactive Publication Date: 2001-07-03
NOVARTIS AG
View PDF0 Cites 12 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

In accordance with yet another aspect of the present invention, there are provided human MMP-8alt antagonists (inhibitors) and methods for identifying such antagonists, wherein such antagonists reduce or prevent the effect of human MMP-8alt polypeptide. Among preferred antagonists are those which mimic human MMP-8alt so as to bind to human MMP-8alt receptor or binding molecules but do not elicit a human MMP-8alt-induced response or more than one human MMP-8alt-induced response. In another embodiment of this aspect of the present invention there are provided antagonists which are small molecules and antibodies and the like which bind to human...

Problems solved by technology

The turnover and remodeling of ECM must be highly regulated since uncontrolled proteolysis contributes to abnormal development and to the generation of many pathological conditions characterized by either excessive degradation or a lack of degradation of ECM components.

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Human neutrophil collagenase splice variant
  • Human neutrophil collagenase splice variant
  • Human neutrophil collagenase splice variant

Examples

Experimental program
Comparison scheme
Effect test

example 1

cDNA and genomic DNA cloning of MMP8alt

Cell culture

Two monocytic cell lines U937 and THP-1 cells were maintained in RPMI-1640 medium with 10% fetal bovine serum (Life Technologies, Inc.). Human chondrocytes were prepared from cartilage taken from patients undergoing joint replacement as described, Aydelotte M. B. and Kuettner K. E., (1988) Connect. Tissue Res. 18:205-222, and cultured in Dulbecco's modified medium with 10% fetal bovine serum.

cDNA and genomic DNA cloning

Total RNA isolation from THP-1 cells, U937 cells and human chondrocytes was accomplished using TriZol reagent (Life Technologies, Inc.). The RNA was reverse transcribed using the 1st Strand cDNA Synthesis Kit (Life Technologies, Inc.) and oligo-dT primer. PCR amplification of MMP-8 cDNA fragment corresponding to nucleotides 68 to 850 of the published sequence (GenBank Accession No. J05556) was performed with Pfu polymerase (Stratagene) for 30 cycles (95.degree. C., 1 min; 55.degree. C., 2 min; 72.degree. C., 3 min) us...

example 2

Assay for In vitro translation and Autoactivation of MMP-8 and MMP-8alt

In vitro translation of MMP-8alt cDNA is subcloned into pCDNA3+ vector is accomplished using TNT T7 Coupled Reticulocyte Lysate System (Promega) with .sup.35 S-methionine (Amersham). In certain cases, canine pancreatic microsomal membranes (Promega) are also included in the in vitro translation reaction to detect co-translational processing and glycosylation. Samples of in vitro translated proteins 50 ml) are diluted to 1 ml with 50 mM Tris-HCl, pH 7.5, 0.2 mM NaCl, 10 mM CaCl.sub.2, and 50 mM ZnCl.sub.2, and concentrated with a Centricon 10 (Amicon) to a final volume of 50 ml. Samples are then diluted 10 fold with the same buffer containing 0.05% Brij-35 and are activated by treatment with 2 mM p-aminophenylmercuric acetate for 90 min at 37.degree. C. In vito translated, .sup.35 S-labeled proteins are subjected to SDS-PAGE and autoradiography. The resultant sample sizes correspond to active MMP-8.

example 3

Assay for identifying MMP-8alt Substrates and Antagonists

An expression cloning procedure is used to identify potential physiological substrates of MMP-8alt. Wen, L.-P. et al., (1997) J. Biol. Chem. 272, 26056-26061; Kothakota, S. et al., (1997) Science 278, 294-298. A chondrocyte cDNA library is prepared in the expression vector pBK-CMV (Statagene). This library is subdivided into small pools in which each pool represents 100 cDNA clones. These pools of cDNA are in vitro transcribed and translated in the presence of .sup.35 S-methionine (Amersham) using the TNT T3 Coupled Reticulocyte Lysate System (Promega). Each translated pool is separated into two parts; one portion is incubated with MMP-8alt, and the other portion is incubated with heat inactivated MMP-8alt. The reaction products are resolved by SDS-PAGE and visualized by autoradiography. Potential substrates are identified by comparing the pattern of .sup.35 S-labeled proteins in the samples treated with active and inactive MM...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

PropertyMeasurementUnit
Fractionaaaaaaaaaa
Fractionaaaaaaaaaa
Compositionaaaaaaaaaa
Login to View More

Abstract

The subject invention is related to human MMP-8alt genes and gene products and their differential expression when comparing a patient with a disease state to a control. A further aspect of the invention concerns compounds which antagonize the biological activity of MMP-8alt protein and methods for identifying these compounds. Another aspect of the present invention concerns pharmaceutical compositions comprising such compounds for the treatment of arthritis, cancer, and disease caused by cellular apoptosis including but not limited to Parkinson's disease, Alzheimer's disease and Huntington's chorea.

Description

BACKGROUNDThe interactions of cells with the extracellular matrix (ECM) are critical for the normal development and function of the organism. Modulation of cell-matrix interactions occurs through the action of unique proteolytic systems responsible for hydrolysis of a variety of ECM components. By regulating the integrity and composition of the ECM structure, these enzyme systems play a pivotal role in the control of signals elicited by matrix molecules, which regulate cell proliferation, differentiation, and cell death. The turnover and remodeling of ECM must be highly regulated since uncontrolled proteolysis contributes to abnormal development and to the generation of many pathological conditions characterized by either excessive degradation or a lack of degradation of ECM components.Matrix metalloproteinases (MMPs) are a major group of enzymes that regulate cell-matrix composition. The MMPs are zinc-dependent endopeptidases known for their ability to cleave one or several ECM con...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C12Q1/68C12N9/64A61K38/00C12Q1/6883
CPCC12N9/6408A61K38/00C12N9/6491C12Q1/6883C12Q2600/158
Inventor HU, SHOU-IH
Owner NOVARTIS AG
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com