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Biomedical subjects

S Zucker

Publications and source records attributed to S Zucker.

At least 55 records · Page 3Linked to original sources
PubMed1994

Mutation of the active site glutamic acid of human gelatinase A: effects on latency, catalysis, and the binding of tissue inhibitor of metalloproteinases-1.

Human gelatinase A, a member of the matrix metalloproteinase family, is secreted from cells as the M(r) 72,000 latent precursor, progelatinase A. The autolytic removal of an N-terminal propeptide generates the M(r) 66,000 active form. Mutants of recombinant progelatinase A, altered such that the proposed active site glutamic acid residue (E375) was replaced by either an aspartic acid (proE375-->D), an alanine (proE375-->A) or a glutamine (proE375-->Q), were purified from medium conditioned by transfected NS0 mouse myeloma cells. Like wild-type progelatinase A, the mutant proenzymes were inactive and could bind tissue inhibitor of metalloproteinases (TIMP)-2 but not TIMP-1 to their C-terminal domains. Their rates of autolytic processing induced by the organomercurial (4-aminophenyl) mercuric acetate, however, were markedly slower and, of the three M(r) 66,000 forms so produced, only E375-->D displayed any proteolytic activity against either a synthetic substrate (kcat/Km = 10% that of the wild-type enzyme) or denatured type I collagen (specific activity = 0.9% that of the wild-type enzyme). ProE375-->A and proE375-->Q could be more rapidly processed to their M(r) 66,000 forms by incubation with a deletion mutant of gelatinase A that has full catalytic activity but lacks the C-terminal domain [delta (418-631) gelatinase A]. These two M(r) 66,000 forms displayed low activity on a gelatin zymogram (approximately 0.01% that of the wild-type enzyme) but, like E375-->D were able to bind TIMP-1 with an affinity equal to that of the activated wild-type enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1994

Comparison of techniques for measurement of gelatinases/type IV collagenases: enzyme-linked immunoassays versus substrate degradation assays.

Radiolabeled substrate degradation assays and gelatin zymography are routinely employed to assay 72 kDa gelatinase A (MMP-2) and 92 kDa gelatinase B (MMP-9) in biological fluids. Enzyme-linked immunosorbent assays (ELISA) have recently been developed for the quantitation of these matrix metalloproteinases (MMP). In this study, we have compared ELISA to standard substrate degradation assays for measurement of MMP-2 and MMP-9 in human plasma and tumor-conditioned media. Gelatin Sepharose chromatography and gel filtration chromatography were employed as partial purification procedures for MMP-2 and MMP-9. The ELISA data for MMP-2 and MMP-9 are linear on a log:log regression curve over a wide range of MMP concentrations and are specific for the designated gelatinase, with no overlap detected with related metalloproteinases. The minimum detectable concentrations of MMP-2 and MMP-9 were approximately 0.5 ng/ml and 0.2 ng/ml, respectively, in the ELISA as compared to 4 ng/ml and 3 ng/ml, respectively, in gelatin zymography. The [3H]gelatin degradation assay required a combination of > 50 ng/ml of MMP-2 and MMP-9 for detection. Although gelatin zymography was less sensitive than ELISA (primarily due to the smaller sample volume employed) and was more difficult to quantitate, this procedure offers the important advantage of being able to distinguish between latent and activated gelatinases.

Collagenases↗
PubMed1994

A measure of closure.

In images of real visual scenes, contours are often fragmented by occlusion, shadows and low reflectance contrast. In order to infer shape from contour, the human visual system must selectively integrate fragments projecting from a common object while keeping fragments from different objects separate. In a previous paper we showed that contour closure has a strong influence on the speed of this process. In this paper we employ a visual search method to evaluate potential measures for the perceptual closure of fragmented shapes. We show that while certain intuitive measures are psychophysically inconsistent, a measure based on a sum of squares of the lengths of contour gaps is appropriate for both polygonal and smooth shapes in that response time can be expressed as a function of this measure. This L2 measure is shown to emphasize large gaps relative to small, and to embody a principle of perceptual regularity.

Discrimination, Psychological↗
PubMed1994

Histologic changes of doxycycline pleurodesis in rabbits. Effect of concentration and pH.

The effects of doxycycline hyclate (DOXY) pleurodesis were compared with those of tetracycline hydrochloride (TCN) in 25 healthy New Zealand White rabbits. One group of rabbits received TCN (pH 2.0) in the left pleural space at a concentration of 35 mg/kg in 3.0 ml of saline solution. Four other groups of rabbits received DOXY in two concentrations (35 mg/kg or 10 mg/kg) and at two pHs (an acid of 2.0 and a NaOH neutralized pH of 7.6). No attempt was made to drain the pleural space after pleurodesis. At autopsy 2 weeks later, bloody fluid filled the pleural cavity and extensive intrapleural adhesions compressed the underlying lung. Microscopically, fibrin and collagen were diffusely deposited in the pleural connective tissue and in the intrapleural adhesions. The visceral pleural thickness increased from its normal value of 0.01 mm to values of 0.5 to 0.9 mm in the TCN and DOXY groups. Fibroblasts were the predominant inflammatory cell in the pleural connective tissue with few neutrophils or lymphocytes. No significant differences of histologic characteristics were observed between the TCN and DOXY groups. Alterations in pH or concentration in the DOXY groups produced similar changes in the gross and microscopic appearance of the pleural space. We conclude that DOXY is as effective as TCN in producing chemical pleurodesis. Doxycycline concentrations of 10 mg/kg were sufficient to substantially sclerose the pleural space in these healthy rabbits. Doxycycline pleurodesis was effective at neutral pH suggesting a mechanism of action independent of acid injury to the mesothelium.

Animals↗
PubMed1994

Elevated plasma stromelysin levels in arthritis.

OBJECTIVE: To determine whether plasma concentrations of stromelysin-1 and gelatinase A are increased in patients with various forms of arthritis. METHODS: A sensitive and specific sandwich enzyme linked immunosorbent assay (ELISA), which employs a murine monoclonal antibody and a rabbit polyclonal antibody to human stromelysin-1, was used to measure plasma stromelysin-1 in 53 healthy subjects, 113 patients with various forms of arthritis and connective tissue diseases, and 65 patients with cancer. Gelatinase A was also measured in these patients using specific polyclonal and monoclonal antibodies to gelatinase A in an ELISA: RESULTS: The plasma concentration of stromelysin-1 (X +/- SEM) was significantly increased (p < 0.001) in patients with rheumatoid arthritis (RA) (187 +/- 14 ng/ml) and systemic lupus erythematosus (SLE) (258 +/- 35 ng/ml) as compared to both healthy control subjects (50 +/- 4 ng/ml) or patients with cancer (61 +/- 20 ng/ml). Plasma stromelysin-1 was also significantly increased in smaller groups of men with osteoarthritis (OA) and gout. In contrast, plasma concentrations of gelatinase A were not significantly increased in patients with RA, OA or gout. In healthy subjects, the concentration of stromelysin-1 was significantly higher in men than women. No correlation was noted between plasma stromelysin-1 levels and age. CONCLUSION: The detection of elevated plasma levels of stromelysin-1 in patients with RA is consistent with increased stromelysin-1 concentrations in inflamed synovial tissues in this disease. The origin of increased plasma stromelysin-1 in SLE is speculative. Measurement of plasma stromelysin-1 may be useful in the diagnosis and management of patients with various forms of arthritis.

Adult↗
PubMed1993

Tumor cell-derived collagenase-stimulatory factor increases expression of interstitial collagenase, stromelysin, and 72-kDa gelatinase.

The tumor cell-derived collagenase-stimulatory factor (TCSF) was previously purified from human lung carcinoma cells (S. M. Ellis, K. Nabeshima, and C. Biswas, Cancer Res., 49: 3385-3391, 1989). This protein is present on the surface of several types of human tumor cells in vitro and in vivo and stimulates production of interstitial collagenase in human fibroblasts. In this study it is shown that TCSF stimulates expression in human fibroblasts of mRNA for stromelysin 1 and 72-kDa gelatinase/type IV collagenase, as well as for interstitial collagenase. Measurement of enzyme protein by immunoassay showed that the amounts of interstitial collagenase and stromelysin 1 were increased in TCSF-treated fibroblasts; gelatin zymography indicated that there was an increase in the 72-kDa gelatinase. These results indicate that tumor cell interaction with fibroblasts via TCSF could lead to increased degradation of interstitial or basement membrane matrix components and thus to enhanced tumor cell invasion.

Animals↗
PubMed1993

Purification and characterization of a novel cytotoxic protein from transformed fibroblasts.

The mechanism by which cancer cells overwhelm normal parenchymal cells during cancer invasion remains obscure. In this article, we describe the purification of a potent cytotoxic protein from plasma membranes of ras oncogene transformed fibroblasts. Tumor cytotoxic protein was purified from detergent extracted tumor membranes by anion exchange and gel filtration chromatography. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate indicated that the hemolytic fractions contained a single protein with an apparent molecular weight of 62,000. A higher concentration of tumor cytotoxic protein was required to lyse fibroblasts as compared to RBC. Based on plasma membrane localization, immunological identity, and biological characteristics, tumor cytotoxic protein is a novel cytolysin which is capable of killing normal cells during cancer invasion.

3T3 Cells↗
PubMed1993

M(r) 92,000 type IV collagenase is increased in plasma of patients with colon cancer and breast cancer.

Overproduction of matrix metalloproteinases (MMPs) is a common characteristic of metastatic cancer cells. Since MMPs can be identified in plasma, we proposed that enhanced MMP-9 secretion by invasive cancer cells may be detected by plasma assay. To this end, we developed a specific sandwich enzyme-linked immunosorbent assay which uses two mouse monoclonal antibodies to human M(r) 92,000 type IV collagenase (MMP-9). The plasma concentration of MMP-9 (mean +/- SD) in 60 healthy subjects (9 +/- 11 ng/ml), 136 patients without cancer, and 179 patients with cancer of the lung, genitourinary tract, or lymphomas-leukemias did not differ significantly. In contrast, plasma MMP-9 was significantly increased (P < 0.01) in 122 patients with gastrointestinal tract cancer and breast cancer (18 +/- 23 and 21 +/- 22 ng/ml, respectively). Whereas carcinoembryonic antigen levels were significantly increased in patients with stage IV gastrointestinal cancer, MMP-9 concentrations were not significantly increased in patients with metastatic disease as compared to those with nonmetastatic cancer. Combining both assays improves sensitivity of detection of colon cancer. MMP-9 was also significantly increased during pregnancy which is consistent with the extensive ongoing tissue remodeling and the leaching of the tissue proteinase into plasma.

Adult↗
PubMed1993

The effect of contour closure on the rapid discrimination of two-dimensional shapes.

An outline drawing often serves as an excellent depiction of a visual scene. Somehow, our visual system can form two- and three-dimensional percepts solely from one-dimensional contour information. In mathematics, contour closure plays a key role in bridging this dimensional gap, however in perception the link between closure and shape is unclear. To better understand this relationship, we devised a set of visual search experiments in which subjects discriminate outline figures by means of their two-dimensional shape. By modulating the degree of closure of the outlines, we show that two-dimensional shape processing is rapid for closed stimuli but slow for open stimuli. We further show that search can be characterized as a smooth, monotonic function of the degree of closure, supporting the notion of a perceptual closure continuum.

Contrast Sensitivity↗
PubMed1993

Tetracycline and doxycycline inhibit pleural fluid metalloproteinases. A possible mechanism for chemical pleurodesis.

We hypothesized that inhibition of matrix-degrading metalloproteinases (MMPs) accounts for a portion of the pleural fibrosis and adhesions of tetracycline pleurodesis. MMPs recently have been described in pleural fluid from patients with both exudative and transudative effusions. Since tetracyclines are recognized inhibitors of other metalloproteinases, we investigated their inhibitory capacity in pleural fluid. High concentrations of several different tetracyclines reduced MMP activity of pleural fluid by more than 75 percent. Lower concentrations (< or = 1 mg/ml) had only modest inhibitory effects. High concentration of of tetracyclines also inhibited cell synthesis of MMPs, in vitro, but other measures of vital cell function were also impaired. We conclude that tetracyclines are effective inhibitors of MMP activity in pleural fluid and may also reduce synthesis of MMPs via non specific cell injury. These data suggest a possible mechanism to account for tetracycline pleurodesis; ie, an inhibition of MMP activity in pleural fluid.

Collagenases↗
PubMed1992

Secretion of gelatinases and tissue inhibitors of metalloproteinases by human lung cancer cell lines and revertant cell lines: not an invariant correlation with metastasis.

Numerous studies have reported a correlation between production of 72-kDa (MMP-2) and 92-kDa (MMP-9) type-IV collagenases/gelatinases and the metastatic potential of cancer cells. An abrogating effect of tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2) on metastases has also been noted. In this report we have used sensitive enzyme-linked immunoassays to measure MMP-2, MMP-9, TIMP-1 and TIMP-2 levels in eight human lung-cancer cell lines which were characterized for biological behavior in nude mice. We demonstrated that the Calu-6 and A549 cell lines with the highest metastatic, invasive and tumorigenic potential secreted the highest levels of MMP-2. MMP-9 and TIMP-1 secretions were comparatively low in all cell lines. TIMP-2 secretion, which exceeded MMP-2 secretion for all cell lines, did not correlate with metastatic potential. To further explore these correlations, the metastatic Calu-6 cell line was transfected with a K-rev-1 cDNA expression construct. The K-rev revertant cell lines demonstrated a more differentiated phenotype and were less tumorigenic, invasive and metastatic in nude mice. Nonetheless, the Calu-6 revertant cell lines secreted higher levels of MMP-2 than the parent cell line. In conclusion, invasion and metastasis by lung-cancer cells requires not only enhanced MMP production, but also other less well-understood tumorigenic characteristics. The multiplicity of factors required by cancer cells for dissemination helps to explain the minute fraction of cancer cells from a primary tumor that ever develop into a metastasis.

Animals↗
PubMed1992

Immunoassay of type IV collagenase/gelatinase (MMP-2) in human plasma.

We have developed a sensitive and specific sandwich type enzyme-linked immunosorbent assay (ELISA) to detect type IV collagenase (MMP-2) in human plasma which employs the combination of affinity purified rabbit polyclonal antibodies and mouse monoclonal antibodies to human MMP-2. The MMP-2 ELISA detects latent and activated MMP-2, MMP-2 complexed with TIMP and MMP-2 complexed with alpha 2 macroglobulin (65% efficiency). To determine whether physiologic conditions associated with increased connective tissue turnover are accompanied by increased MMP-2 levels in plasma, we compared enzyme levels in pregnant and nonpregnant women. Plasma MMP-2 (mean +/- standard deviation) was significantly increased (p less than 0.05) in the second half of pregnancy (650 +/- 312) as compared to early pregnancy (356 +/- 139) or the nonpregnant state (387 +/- 193). As a result of the linkage between type IV collagenase production by cancer cells and the metastatic phenotype, the assay of MMP-2 in plasma is of potential clinical value in cancer.

Antibodies, Monoclonal↗
PubMed1992

Modulation of the antigenic phenotype of human melanoma cells by differentiation-inducing and growth-suppressing agents.

Tumor cells often display alterations in their normal program of cellular differentiation. A promising approach for the treatment of cancer involves the induction of terminal differentiation and a loss of proliferative capacity in cancer cells. In human melanoma cells, the combination of mezerein (MEZ) and fibroblast interferon (IFN-beta), results in a rapid and irreversible suppression of cell growth with a concomitant increase in the synthesis of melanin. The induction of terminal differentiation is associated with alterations in the expression of several cellular genes, including fibronectin, ISG-15 and ISG-54, and changes in the expression of specific cell surface antigens, including intercellular adhesion molecule-1 (ICAM-1) and HLA Class I antigens. In the HO-1 human melanoma cell line, induction of terminal differentiation by MEZ plus IFN-beta results in an induction and/or increased expression of ICAM-1, HLA Class I antigens and HLA Class II antigens. IFN-beta and MEZ alone can modulate expression of these antigens to a lower extent than does the combination of compounds. Induction of terminal differentiation and the irreversible suppression of cell growth is not a prerequisite for antigenic modulation in HO-1 cells. This is indicated by the inability of immune interferon (IFN-gamma), a strong inducer of ICAM-1, HLA Class I antigens and HLA Class II antigens synthesis, or the combination of IFN-beta plus IFN-gamma which synergistically but reversibly suppresses HO-1 growth, to induce melanin synthesis or terminal differentiation in HO-1 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1992

Human pleural effusions are rich in matrix metalloproteinases.

We identified and characterized type IV collagenase and gelatinase activity in pleural fluid from 32 patients. The capacity to substantially degrade type IV collagen was demonstrated in every pleural sample. Comparable results were also noted for the degradation of a radiolabeled gelatin substrate. Gelatin gel zymography of the pleural fluids revealed two prominent zones of lysis at 66 kDa and 92 kDa. These were identified by specific polyclonal antibodies as human matrix metalloproteinases MMP-2 and MMP-9. The concentration of MMP-2 in pleural fluid, as measured by enzyme-linked immunoassay, averaged 1,622 ng/ml whereas those of MMP-9 were 210 ng/ml. Substrate degradation activity was compared in both serum and pleural fluid from three patients and found to be similar. In serum this enzymatic activity was primarily due to MMP-9 whereas in pleural fluid, the predominant gelatinase was MMP-2. This was confirmed by immunoassay that showed that MMP-2 levels were two to five times higher in pleural fluid than in serum. We conclude that substantial amounts of MMP-2 and, to a lesser degree, MMP-9 are present in pleural effusions. The bioactivity and the immunoactivity of these enzymes did not help to distinguish among pleural fluids characterized as transudates, nonmalignant exudates, or malignant exudates. The differences in the distribution of these enzymes in pleural fluid and blood suggest that their presence is not due simply to the ultrafiltration of plasma, but rather to synthesis by the resident cells at the pleural surfaces.

Biomarkers↗
PubMed1992

Characterization of metalloproteinases and tissue inhibitors of metalloproteinases in human plasma.

In this study, we have identified and characterized metalloproteinases and tissue inhibitors of metalloproteinases (TIMPs) in human plasma. Treatment of plasma with trypsin or aminophenylmercuric acetate resulted in activation of latent gelatinolytic activity. Fractionation of plasma by gelatin Sepharose chromatography resulted in the isolation of 72 kDa and 92 kDa gelatinases/type IV collagenases. The 72 kDa gelatinase was purified by gel filtration chromatography. Stromelysin-1 was isolated from plasma by Matrex green A affinity chromatography. Immunoblotting of plasma fractions with antibodies to unique peptide regions of human gelatinases differentiated the 72 kDa gelatinase from the 92 kDa gelatinase. Antibodies to the amino terminal peptides of each enzyme were used to determine that plasma gelatinases circulate as latent proenzymes. Immunoblotting with antibodies directed against human stromelysin identified a 57 kDa stromelysin. TIMP-1 (28 kDa) and TIMP-2 (21 kDa) were also identified by immunoblotting of gelatin Sepharose bound plasma proteins using non-crossreacting antibodies to each protein.

Amino Acid Sequence↗
PubMed1992

Growth inhibition and modulation of antigenic phenotype in human melanoma and glioblastoma multiforme cells by caffeic acid phenethyl ester (CAPE)

The active component of the honeybee hive product propolis, caffeic acid phenethyl ester (CAPE), has been shown to display increased toxicity toward various oncogene-transformed cell lines in comparison with their untransformed counterparts (Su et al., 4: 231-242, 1991). This observation provides support for the concept that it is the transformed phenotype which is specifically sensitive to CAPE. In the present study, we have determined the effect of CAPE on the growth and antigenic phenotype of a human melanoma cell line, HO-1, and a human glioblastoma multiforme cell line, GBM-18. For comparison, we have also tested the effects of mezerein (MEZ), mycophenolic acid (MPA) and retinoic acid (RA), which can differentially modulate growth, differentiation and the antigenic phenotype in these human tumor cell lines. Growth of both cell lines was suppressed by CAPE in a dose-dependent fashion, with HO-1 cells being more sensitive than GBM-18 cells. The antiproliferative effect of CAPE was enhanced in both cell types if CAPE and MEZ were used in combination. Growth suppression was associated with morphological changes in H0-1 cells, suggesting induction of a more differentiated phenotype. CAPE also differentially modulated the expression of several antigens on the surface of the two tumor cell lines. These results suggest a potential role for CAPE as an antitumor agent, an antigenic modulating agent and possibly a differentiation inducing agent.

Antigens, Neoplasm↗