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PubMed2001

Particulate dermal matrix as an injectable soft tissue replacement material.

Products currently used as injectable soft tissue replacement materials in the dermatologic, plastic and reconstructive, and urological fields exhibit several shortfalls including reactivity, migration, rapid degradation, and necessity of a donor site. This study examines the feasibility of providing a particulate acellular human dermal matrix for injection as a soft tissue replacement material that addresses many of these issues. Animal feasibility studies tested differences in implant performance related to processing techniques, matrix concentration, and volume of the collagen matrix to be injected. Results demonstrated that processing techniques that involve shearing and tearing of the dermal collagen matrix resulted in frayed and damaged collagen bundles and led to rapid resorption or loss of the implant, when injected subcutaneously in a rat model. Processing the collagen matrix in liquid nitrogen resulted in less damage to the collagen matrix and exhibited longer persistence, when compared to the damaged collagen matrix. This particulate matrix also exhibits rapid repopulation by host cells that should enhance revascularization and remodeling. The particulate nature of this processed dermal matrix allows for easy delivery of concentrations up to 330 mg/mL, which exceeds that of other currently used products. This increased concentration should allow for decreased need of "overcorrection" and repeated injections.

Animals↗
PubMed1994

Tartrate-resistant acid phosphatase accumulated in the matrix of developing medullary bone induced by estrogen treatment of male Japanese quail.

Tartrate-resistant acid phosphatase (TRAP) in medullary bone matrix and bone cells was histochemically examined employing estrogen-induced medullary bone of male Japanese quail. Within 2 days after estrogen administration, medullary bone matrix was identified and the matrix and osteoblasts were negative for TRAP activity. Osteoclasts showing TRAP activity were occasionally seen. By 4 days, the projection of medullary bone trabeculae was seen and TRAP became positive in the deeper part of the bone matrix of trabeculae as well as the matrix located near cortical bone, but the surface areas and extending margins of medullary bone trabeculae were negative in activity. Osteoblasts were also negative for TRAP activity. By 7 days, TRAP-positive bone matrix conspicuously increased accompanying increment of bone volume, but TRAP-negative bone matrix was still seen at the surface areas or extending areas of the medullary bone trabeculae. Osteoblasts showed no reaction product. Osteoclasts showing TRAP activity increased in number. The results demonstrate that TRAP accumulates in medullary bone matrix with bone formation. Circumstantial evidence suggests that medullary bone osteoblasts may be involved in TRAP accumulation in the matrix despite their negative activity.

Acid Phosphatase↗
PubMed1995

Mineralization of bone-like extracellular matrix in the absence of functional osteoblasts.

When grown in medium containing ascorbic acid and beta-glycerol phosphate, mouse MC3T3-E1 cells express an osteoblast phenotype and produce a highly mineralized extracellular matrix. The purpose of this study was to independently examine the role of the collagenous matrix and functional osteoblasts on the mineralization process. Cultures with and without an extensive collagenous matrix were prepared by growing MC3T3-E1 cells in the presence and absence of ascorbic acid. Matrix-rich cultures mineralized at much lower calcium phosphate ion products than nonmatrix cultures. At higher ion products, spontaneous precipitation in the medium and cell layers of nonmatrix cultures were observed. In contrast, mineral in matrix-rich cultures was still exclusively associated with collagen fibrils and not with ectopic sites in the cell layer or medium. To examine the effect of cell viability on matrix mineralization, cells were grown 8 or 16 days in the presence of ascorbic acid, then killed and incubated in a mineralizing medium. Significant mineralization was not observed in the collagenous matrix of 8-day killed cultures or age-matched controls. At 16 days mineral was associated with collagen fibrils at specific foci in the matrix of both viable and killed cultures. This observation is consistent with the concept that collagenous matrices must undergo a maturation process before they can support a mineral induction and growth. It further shows that osteoblast-like cells are not required for mineralization of mature matrices, but are required for matrix maturation.

3T3 Cells↗
PubMed2002

Heat shock mediated modulation of protein kinase CK2 in the nuclear matrix.

Nuclear matrix, a key structure in the nuclear framework, appears to be a particularly responsive target during heat shock treatment of cells. We have previously shown that nuclear matrix is a preferential target for protein kinase CK2 signaling in the nucleus. The levels of CK2 in the nuclear matrix undergo dynamic changes in response to altered growth status in the cell. Here, we have demonstrated that CK2 targeting to the nuclear matrix is profoundly influenced by treatment of the cells to temperatures higher than 37 degrees C. Rapid increase in the nuclear matrix association of CK2 is observed when cells are placed at temperatures of 41 and 45 degrees C. This effect at 45 degrees C was higher than at 41 degrees C, and was time-dependent. Also, different cell lines behaved in a qualitatively similar manner though the quantitative responses differed. The modulations in the nuclear matrix associated CK2 in response to heat shock appear to be due to trafficking of the enzyme between cytosolic and nuclear compartments. In addition, it was noted that isolated nuclei subjected to heat shock also responded by a shuttling of the intrinsic CK2 to the nuclear matrix compartment. These results suggest that modulations in CK2 in the nuclear compartment in response to the heat stress occur not only by a translocation of the enzyme from the cytoplasmic compartment to the nuclear compartment, but also that there is a redistribution of the kinase within the nuclear compartment resulting in a preferential association with the nuclear matrix. The results support the notion that CK2 association with the nuclear matrix in response to heat shock may serve a protective role in the cell response to stress.

Casein Kinase II↗
PubMed2005

RNA-dependent nuclear matrix contains a 33 kb globin full domain transcript as well as prosomes but no 26S proteasomes.

Previously, we have shown that in murine myoblasts prosomes are constituents of the nuclear matrix; a major part of the latter was found to be RNase sensitive. Here, we further define the RNA-dependent matrix in avian erythroblastosis virus (AEV) transformed erythroid cells in relation to its structure, presence of specific RNA, prosomes and/or proteasomes. These cells transcribe but do not express globin genes prior to induction. Electron micrographs show little difference in matrices treated with DNase alone or with both, DNase and RNase. In situ hybridization with alpha globin riboprobes shows that this matrix includes globin transcripts. Of particular interest is that, apparently, a nearly 35 kb long globin full domain transcript (FDT), including genes, intergenic regions and a large upstream domain is a part of the RNA-dependent nuclear matrix. The 23K-type of prosomes, previously shown to be co-localized with globin transcripts in the nuclear RNA processing centers, were found all over the nuclear matrix. Other types of prosomes show different distributions in the intact cell but similar distribution patterns on the matrix. Globin transcripts and at least 80% of prosomes disappear from matrices upon RNase treatment. Interestingly, the 19S proteasome modulator complex is insensitive to RNase treatment. Only 20S prosomes but not 26S proteasomes are thus part of the RNA-dependent nuclear matrix. We suggest that giant pre-mRNA and FDTs in processing, aligning prosomes and other RNA-binding proteins are involved in the organization of the dynamic nuclear matrix. It is proposed that the putative function of RNA within the nuclear matrix and, thus, the nuclear dynamic architecture, might explain the giant size and complex organization of primary transcripts and their introns.

Base Sequence↗
PubMed1989

Effects of interleukin-1 on fibroblast extracellular matrix, using a 3-dimensional culture system.

This study describes the alterations induced by Interleukin-1 alpha and -beta (IL-1 alpha and IL-1 beta) on fibroblast-synthesized extracellular matrix. Fibroblasts were grown between pieces of dentin or in collagen-coated Terasaki wells for 3 or 6-9 weeks to create 3-dimensional cell-containing matrices constituted primarily of proteoglycans and collagens, respectively. Following incubation with IL-1 alpha or IL-1 beta (10(-9) M) at 37 degrees C for 24 or 72 hr, samples were prepared for light and electron microscopy. Both IL-1 alpha and IL-1 beta induced collapse of the extracellular matrix by 72 hr, as manifested by a decrease of the cross-sectional area and an increased density of the matrices. Three-week matrices were reduced 26% and 45% by using IL-1 alpha and IL-1 beta, respectively. Comparable values obtained by using 6-week matrices were 14% and 30%. Cells within the matrix, normally stellate in shape with numerous extended processes, attained a more rounded or spindle shape with few and reduced processes and showed apparent alterations at cell matrix attachment sites and rearrangement of the cytoskeleton. Elongated cells at the top of the matrix appeared more compressed. The alterations were more pronounced in cultures incubated with IL-beta than with IL-1 alpha. Immunocytochemistry of extracellular matrix components revealed a decrease in staining intensity of chondroitin and dermatan sulfate in the 3-week matrix following IL-1 beta incubation. There was also a decrease in collagen type 1 staining of 9-week matrices treated with IL-1 alpha or IL-1 beta. These studies show that IL-1 has an effect on fibroblast-synthesized extracellular matrix and indicate that the effects of IL-1 alpha and IL-1 beta may differ. The resulting collapse of the matrix appears at least in part to be due to changes in proteoglycans and collagens.

Actin Cytoskeleton↗
PubMed1998

Effects of injurious compression on matrix turnover around individual cells in calf articular cartilage explants.

The effects of mechanical injury on the metabolism of cartilage matrix are of interest for understanding the pathogenesis of osteoarthrosis and the development of strategies for cartilage repair. The purpose of the present study was to examine the effects of injury on matrix turnover in a calf articular cartilage explant system for which the effects of mechanical loading on cell activity and the cell-mediated pathways of matrix metabolism are already well characterized. New methods of quantitative autoradiography were used in combination with established biochemical and biomechanical techniques for the analysis of cell and matrix responses to acute mechanical injury, with particular attention to the processes of localized matrix turnover in the cell-associated matrices of individual chondrocytes. Matrix deposition and turnover around cells in control explants was spatially dependent, with the highest rates of proteoglycan deposition and turnover and the lowest rates of collagen deposition (as indicated by [3H]proline autoradiography) occurring in the pericellular matrix. Injurious compression was associated with (a) an abrupt decrease in the tensile load-carrying capacity of the collagen matrix, apparently associated with mechanical failure of the tissue, (b) a considerable but subtotal decrease in cell viability, marked by the emergence of an apparently inactive cell population interspersed within catabolically active but abnormally large cells, and (c) sustained, elevated rates of proteoglycan turnover, particularly in the cell-associated matrices of apparently viable cells, which involved the increased release of aggregating species in addition to a spectrum of degradation fragments that were also in controls. These results may represent an in vitro model for the responses of chondrocytes and the cartilage extracellular matrix to mechanical injury.

Animals↗
PubMed1999

Increased expression of matrix metalloproteinase-1 in osteocytes precedes bone resorption as stimulated by disuse: evidence for autoregulation of the cell's mechanical environment?

An in vivo animal model of bone adaptation was used to examine a possible role for matrix metalloproteinase-1 in the local mediation of bone remodeling: to corrode the coupling of osteocytes to the matrix in an attempt to autoregulate the cell's perception of its mechanical environment. Twelve young (12-16 months old) skeletally mature turkeys were separated into groups to be studied for stimulus periods of either 3 or 30 days. In each animal, the left ulna was functionally isolated and subjected to either disuse or 3,000 microstrain at 1 Hz for 10 minutes per day. The right ulna remained intact and served as an intra-animal control. No significant differences in bone area were detected at 3 days; however, ulnae subjected to disuse lost 8 +/- 4% (+/-SD) of bone area by 30 days. Over the same period, ulnae subjected to the mechanical stimulus gained 21 +/- 9% of bone area. With use of in situ reverse transcription-polymerase chain reaction, less than 2% of the osteocytes examined from the intact control ulnae stained positively for matrix metalloproteinase-1 mRNA. An antibody raised against matrix metalloproteinase-1 revealed no positively labeled osteocytes in the intact ulnae. This low percentage of osteocytes expressing matrix metalloproteinase-1 mRNA was similar to that seen in ulnae subjected to the osteogenic mechanical stimuli. In contrast, ulnae subjected to either 3 or 30 days of disuse showed evidence of matrix metalloproteinase-1 mRNA activity in a high percentage of osteocytes (89 +/- 5 and 66 +/- 8%, respectively; each time point significantly different from intact ulnae, as well as from each other, p < 0.05). The percentage of osteocytes labeled with the anti-matrix metalloproteinase-1 antibody was also highly elevated following 3 days of disuse (74 +/- 17%). These data demonstrate that an early response of bone to disuse is the upregulation of matrix metalloproteinase-1 activity in osteocytes. It is proposed that this upregulation of collagenase activity is indicative of the cell's degradation of coupling to the matrix, and it thus reflects the osteocyte's regulation of its own mechanical environment. We believe that such autoregulation of the osteocyte's physical environment will accommodate subtle changes in the bone's functional environment without the need to add or resorb bone tissue.

Animals↗
PubMed2005

Identification of novel lysosomal matrix proteins by proteome analysis.

The lysosomal matrix is estimated to contain about 50 different proteins. Most of the matrix proteins are acid hydrolases that depend on mannose 6-phosphate receptors (MPR) for targeting to lysosomes. Here, we describe a comprehensive proteome analysis of MPR-binding proteins from mouse. Mouse embryonic fibroblasts defective in both MPR (MPR 46-/- and MPR 300-/-) are known to secrete the lysosomal matrix proteins. Secretions of these cells were affinity purified using an affinity matrix derivatized with MPR46 and MPR300. In the protein fraction bound to the affinity matrix and eluted with mannose 6-phosphate, 34 known lysosomal matrix proteins, 4 candidate proteins of the lysosomal matrix and 4 non-lysosomal contaminants were identified by mass spectrometry after separation by two-dimensional gel electrophoresis or by multidimensional protein identification technology. For 3 of the candidate proteins, mammalian ependymin-related protein-2 (MERP-2), retinoid-inducible serine carboxypeptidase (RISC) and the hypothetical 66.3-kDa protein we could verify that C-terminally tagged forms bound in an M6P-dependent manner to an MPR-affinity matrix and were internalized via MPR-mediated endocytosis. Hence these 3 proteins are likely to represent hitherto unrecognized lysosomal matrix proteins.

Animals↗
PubMed1994

Metalloproteinase-dependent neurite outgrowth within a synthetic extracellular matrix is induced by nerve growth factor.

In order to assess the requirement for matrix metalloproteinases in neuronal regeneration, in vitro neurite outgrowth by chick dorsal root ganglionic neurons (DRGn) was examined within a reconstituted extracellular matrix. For these studies, cultured neurons were treated with a synthetic peptide inhibitor of metalloproteinases (spIMP), LMHKPRCGVPDVGG. spIMP inhibited all neuronal metalloproteinase activities in zymography and substrate-release assays and was used to examine the role of metalloproteinases in neurite outgrowth by DRGn. Cultures of dissociated DRGn rapidly extended neurites on planar extracellular matrix substrates and this rate of outgrowth was not affected by adding NGF or spIMP. In contrast, neurite extension within a three-dimensional gel of extracellular matrix increased nearly threefold after adding NGF. The NGF-induced neurite penetration was negated in the presence of spIMP but not by control peptide. Similar results were obtained using explanted dorsal root ganglia. These findings suggested that NGF-induced neurite outgrowth within an extracellular matrix involves metalloproteinase activity. Zymographic analysis of media conditioned by NGF-treated DRGn revealed a pair of gelatinolytic bands with apparent molecular masses 72 and 66 kDa, which comigrated as a single 66-kDa band after activation with an organomercurial agent. The gelatinase activities were calcium- and zinc-dependent and were absent from zymograms developed in the presence of spIMP, indicating that NGF-treated DRGn release and activate a 72-kDa metalloproteinase. Samples from DRGn cultures treated with low levels of NGF contained similar amounts of latent and activated metalloproteinase, while high levels of NGF induced an apparent increase in total metalloproteinase secretion and a substantially greater proportion of activated enzyme. Western blot analysis showed this metalloproteinase was immunologically similar to 72-kDa type IV collagenase and immunoassays revealed that this matrix metalloproteinase was increased threefold by high NGF. Furthermore, after high NGF treatment, DRGn media contained sixfold more metalloproteinase activity in assays of matrix degradation. In summary, these results indicate that NGF enhanced metalloproteinase-dependent neurite outgrowth of DRGn within a reconstituted extracellular matrix. Also, NGF increased the expression and activation of 72-kDa type IV collagenase, suggesting a role for this matrix-degrading metalloproteinase in neuronal regeneration.

Amino Acid Sequence↗
PubMed1996

Extracellular matrix collagen synthesis and degradation following coronary balloon angioplasty.

Percutaneous transluminal coronary angioplasty is associated with intimal hyperplasia and extracellular matrix deposition of collagen, leading to restenosis in a significant number of cases. The purpose of the present study was to determine the effects of balloon angioplasty on extracellular matrix collagen content and collagenase activity in a porcine coronary artery restenosis model 6 weeks following balloon injury. We tested the hypothesis that in balloon-injured arteries the neointimal extracellular matrix was characterized by increased collagen content and decreased metalloproteinase activity relative to non-injured arteries. Male miniswine maintained on a high cholesterol diet underwent cardiac catheterization and double balloon injury to the right and left circumflex coronary arteries. The coronary arteries were either pressure-perfusion-fixed and prepared for histological examination, or dissected free of adventitia for further collagen and matrix metalloproteinase studies. Collagen synthesis in balloon-injured coronary arteries was compared to non-injured arteries using Northern blot analysis and histochemical stains. Comparative studies on differences between balloon-injured and non-balloon-injured arterial matrix metalloproteinase activity were done using zymography. Balloon angioplasty arterial injury resulted in a significant increase in type I collagen mRNA expression, with increased collagen deposition in the extracellular matrix. In contrast, matrix metalloproteinase activity was markedly decreased. The results suggest that the increased neointimal extracellular matrix observed late in the injury response may be due to not only increased collagen synthesis, but also reduced degradation. The failure to achieve a balance between the synthesis and degradation of extracellular matrix collagen could serve as an important mechanism responsible for restenosis.

Angioplasty, Balloon, Coronary↗
PubMed2001

The conserved domain CR2 of Epstein-Barr virus nuclear antigen leader protein is responsible not only for nuclear matrix association but also for nuclear localization.

There is a growing body of evidence for the importance of the nuclear matrix in various nuclear events including gene expression and DNA replication. Epstein-Barr virus (EBV) nuclear antigen leader protein (EBNA-LP) is a nuclear matrix-associated protein that has been suggested to play an important role in EBV-induced transformation. To define the biological significance of the association of EBNA-LP with the nuclear matrix, we mapped the domain of EBNA-LP responsible for nuclear matrix association and investigated the functions of the EBNA-LP mutant mutagenized by substitution of alanines for the cluster of arginine residues in the mapped region. The results of the present study were as follows. (i) Transiently expressed EBNA-LP in COS-7 or BOSC23 cells was associated with the nuclear matrix, similarly to that in EBV-infected B cells. (ii) Mutational analysis of EBNA-LP revealed that a 10-amino acid segment of EBNA-LP is critical for nuclear matrix association of the protein. Interestingly, the identified region overlapped with the region CR2 of EBNA-LP conserved among a subset of primate gammaherpesviruses. The identified segment is referred to as EBNA-LP NMTS (nuclear matrix targeting signal). (iii) The EBNA-LP mutant with the arginine to alanine substitutions in NMTS was no longer localized not only to the nuclear matrix but also to the nucleus. (iv) The EBNA-LP mutant lacked its ability to coactivate EBNA-2-dependent transactivation. These results indicated that EBNA-LP needs to be localized in the nucleus and/or associated with the nuclear matrix through CR2 to elicit its function such as the coactivation of the EBNA-2-dependent transcriptional activation.

Alanine↗
PubMed1996

Bone cell matrix promotes the adhesion of human prostatic carcinoma cells via the alpha 2 beta 1 integrin.

Prostatic carcinoma cells have a propensity to metastasize to bone, and we propose that this phenomenon may be promoted by the adhesion of metastatic cells to bone matrix. Bone matrix is produced by osteoblasts, and we have developed an in vitro model of bone matrix by isolating the substratum deposited by human osteoblast-like U2OS cells. The collagenous nature of this matrix was demonstrated by the incorporation of [3H]proline and its subsequent release by purified collagenase. Both U2OS matrix and purified type I collagen stimulated the adhesion of human PC-3 prostatic carcinoma cells. Human laminin supported adhesion to a much lesser extent, and PC-3 cells did not adhere to fibronectin. Adhesion of PC-3 cells to U2OS matrix closely resembled adhesion to purified type I collagen with respect to (a) inhibition by a collagen-derived peptide and by antibodies raised against alpha 2 or beta 1 integrin collagen receptor subunits; (b) lack of inhibition by RGD (Arg-Gly-Asp) peptides; (c) stimulation by Mn2+ and Mg2+ ions but not by Ca2+ ion; and (d) stimulation by the phorbol ester PMA (phorbol 12-myristate 13-acetate). This adhesion was also stimulated (2.3-fold) by transforming growth factor beta (TGF-beta), which is a major bone-derived growth factor. We conclude that human osteoblast-like matrix is an adhesive substrate for PC-3 prostate carcinoma cells. This adhesion appears to be mediated by the interaction of alpha 2 beta 1 integrin on PC-3 cells with matrix-derived collagen. The stimulation of this adhesion by TGF-beta suggests that the co-expression of TGF-beta and type I collagen in bone may synergistically facilitate the adhesion of metastatic cells to bone matrix proteins and thereby increase their localization in the skeleton.

Amino Acid Sequence↗
PubMed1992

The extracellular matrix of cartilage in the growth plate before and during calcification: changes in composition and degradation of type II collagen.

Calcification occurs in the extracellular matrix of the hypertrophic zone of the growth plate when the extracellular matrix volume is reduced to a minimum and alkaline phosphatase content is maximal. The present study shows that significant quantitative and qualitative changes occur in the composition and structure of macromolecules in the extracellular matrix before and during calcification in the proximal tibial growth plate of the bovine fetus. These were detected in part by using microchemical and microimmuno-chemical analyses of sequential transverse frozen sections at defined sites throughout the growth plate. Concentrations of matrix molecules in the extracellular matrix have not previously been determined biochemically. They were measured per unit matrix volume by using combined immunochemical/chemical-histomorphometric analyses. The concentrations within the extracellular matrix of the C-propeptide of type II collagen, aggregating proteoglycan (aggrecan), and hyaluronic acid all progressively increased in the maturing and hypertrophic zones, being maximal (or near maximal) at the time of initiation of mineralization. These results for proteoglycan are contrary to some earlier reports of a loss of proteoglycan are contrary to some earlier reports of a loss of proteoglycan prior to mineralization which measured the tissue content of proteoglycan rather than that present in the extracellular matrix, the volume of which is progressively reduced as the growth plate matures. The C-propeptide data provides a quantitative confirmation of previous immunohistochemical studies. Total collagen concentration (measured as hydroxyproline) in the extracellular matrix initially increased through the proliferating and maturing zones but then rapidly decreased in the hypertrophic zone.(ABSTRACT TRUNCATED AT 250 WORDS)

Acid Phosphatase↗
PubMed1999

Host expression of matrix metalloproteinase-2 and tissue inhibitor of metalloproteinase-2 in normal colon tissue affects metastatic potential of colorectal cancer.

PURPOSE: To clarify the mechanism of cancer cell invasion, we paid close attention to the role of matrix metalloproteinases and tissue inhibitors of metalloproteinases in normal tissue that is located in the same organ as the cancer. METHODS: Samples were obtained from a tumor lesion and normal tissue in the resected large intestine of 59 patients with colorectal cancer, including 13 cases with liver metastasis (Group A) and 46 cases without liver metastasis (Group B). In each sample the expression of m-RNA for matrix metalloproteinase-2, matrix metalloproteinase-9, tissue inhibitor of metalloproteinase-1, and tissue inhibitor of metalloproteinase-2 was examined using reverse transcription-coupled polymerase chain reaction and southern hybridization. RESULTS: In normal colon tissue the expression rate of matrix metalloproteinase-2 in Group A (76.9 percent) was significantly higher than that of Group B (15.2 percent; P < 0.0001). Regarding the expression pattern of m-RNA of matrix metalloproteinase-2 and tissue inhibitor of metalloproteinase-2 in normal colon tissue, Group B included 24 cases with matrix metalloproteinase-2 negative, and tissue inhibitor of metalloproteinase-2 positive (24/46; 52.2 percent). Conversely, Group A had only one case with matrix metalloproteinase-2 negative and tissue inhibitor of metalloproteinase-2 positive (1/13; 7.7 percent; P = 0.0107). In addition, the ratio of cases with matrix metalloproteinase-2 positive and tissue inhibitor of metalloproteinase-2 negative in Group A was 30.8 percent (4/13), which was a significantly higher rate than that in Group B (3/46; 6.5 percent; P = 0.0170). CONCLUSION: We think that the expression pattern of m-RNA of matrix metalloproteinase-2 and tissue inhibitor of metalloproteinase-2 in normal colon tissue is closely related to liver metastasis in colon cancer patients.

Aged↗
PubMed2005

Pyridinium-based ionic liquid matrices can improve the identification of proteins by peptide mass-fingerprint analysis with matrix-assisted laser desorption/ionization mass spectrometry.

Matrix-assisted laser desorption/ionization mass spectrometry has become an indispensable tool for identification of proteins by peptide mass-fingerprint analysis. Selection of the matrix, addition of matrix additives, and sample-preparation techniques are known to affect the quality of the spectra and hence protein identification. We investigated the effect of pyridine as matrix additive for the commonly used crystalline matrix alpha-cyano-4-hydroxycinnamic acid (CCA), forming a pyridinium based ionic liquid matrix, on the mass spectra of synthetic peptides and tryptic protein digests. Beside the equimolar mixture of CCA and pyridine, the effect of addition of substoichiometric amounts of the base to the acid was tested. Optimum results in terms of signal-to-noise ratios, reduction of chemical noise, and reduced formation of alkali adducts and matrix clusters were observed for the matrix CCA-pyridine in the molar ratio 2:1. The optimized ionic liquid matrix was used for identification of tryptic digests of six model proteins and for identification of a protein extracted from a two-dimensional gel with the proteome of the bacterium Corynebacterium glutamicum, and shown to facilitate protein identification, yielding higher scores and increased sequence coverage compared with pure CCA. Thus CCA-Py 2:1 is a potential alternative for identification and characterization of proteins by peptide mass-fingerprint analysis.

Corynebacterium glutamicum↗
PubMed1996

Vitamin D metabolites regulate matrix vesicle metalloproteinase content in a cell maturation-dependent manner.

Matrix vesicles are extracellular organelles produced by cells that mineralize their matrix. They contain enzymes that are associated with calcification and are regulated by vitamin D metabolites in a cell maturation-dependent manner. Matrix vesicles also contain metalloproteinases that degrade proteoglycans, macromolecules known to inhibit calcification in vitro, as well as plasminogen activator, a proteinase postulated to play a role in activation of latent TGF-beta. In the present study, we examined whether matrix vesicle metalloproteinase and plasminogen activator are regulated by 1, 25(OH)2D3 and 24,25(OH)2D3. Matrix vesicles and plasma membranes were isolated from fourth passage cultures of resting zone chondrocytes that had been incubated with 10(-10)-10(-7) M24, 25(OH)2D3 or growth zone chondrocytes incubated with 10(-11)-10(-8) M 1,25(OH)2D3, and their alkaline phosphatase, active and total neutral metalloproteinase, and plasminogen activator activities determined. 24,25(OH)2D3 increased alkaline phosphatase by 35-60%, decreased active and total metalloproteinase by 75%, and increased plasminogen activator by fivefold in matrix vesicles from resting zone chondrocyte cultures. No effect of vitamin D treatment was observed in plasma membranes isolated from these cultures. In contrast, 1,25(OH)2D3 increased alkaline phosphatase by 35-60%, but increased active and total metalloproteinase three- to fivefold and decreased plasminogen activator by as much as 75% in matrix vesicles isolated from growth zone chondrocyte cultures. Vitamin D treatment had no effect on plasma membrane alkaline phosphatase or metalloproteinase, but decreased plasminogen activator activity. The results demonstrate that neutral metalloproteinase and plasminogen activator activity in matrix vesicles are regulated by vitamin D metabolites in a cell maturation-specific manner. In addition, they support the hypothesis that 1,25(OH)2D3 regulation of matrix vesicle function facilitates calcification by increasing alkaline phosphatase and phospholipase A2 specific activities as well as metalloproteinases which degrade proteoglycans.

24,25-Dihydroxyvitamin D 3↗
PubMed2000

A new look at the nuclear matrix.

The concept of the nuclear matrix, a karyoskeletal structure that serves as a support for the genome and its activities, has stimulated many studies of the association of nuclear components and functions with this structure. However, certain experimental findings are not consistent with the existence of the nuclear matrix in vivo, including our inability to visualise a corresponding structure in intact cells, the demonstrated mobility in vivo of chromatin and messenger ribonucleoprotein particles, which are claimed to be bound to the nuclear matrix, the paradoxical extractability from nuclei in low ionic strength buffers of enzymes that are found in the 2 M NaCl-insoluble matrix, and the extractability, in conditions which reproduce the intranuclear milieu, of regions of DNA (matrix or scaffold attachment regions, MAR/SARs) postulated to be bound to the nuclear matrix in vivo. This review considers the nuclear matrix model in the light of sometimes overlooked evidence that each step in its isolation may cause nuclear components to bind to it by new liaisons that do not exist in vivo. This is illustrated by experiments where nuclear-targeted green fluorescent protein is found in the nuclear matrix, and raises the possibility that MAR/SARs actually bind to DNA-binding proteins or multiprotein complexes, including replicational, transcriptional and processing machinery, and topoisomerases that are incorporated into the nuclear matrix during its preparation. Considering that the nuclear lamina forms a rigid exoskeleton, the necessity for internal skeletal structures is raised; the major roles that macromolecular crowding, phase partitioning, and charge effects are likely to play in organisation of the intranuclear space may provide new models for the compartmentalisation of proteins and functions into different nuclear domains and of chromosomes into territories.

DNA↗