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M J Banda

Publications and source records attributed to M J Banda.

At least 19 recordsLinked to original sources
PubMed1992

Large induction of keratinocyte growth factor expression in the dermis during wound healing.

Recent studies have shown that application of basic fibroblast growth factor (basic FGF) to a wound has a beneficial effect. However, it has not been assessed whether endogenous FGF also plays a role in tissue repair. In this study we found a 160-fold induction of mRNA encoding keratinocyte growth factor (KGF) 1 day after skin injury. This large induction was unique within the family of FGFs, since mRNA levels of acidic FGF, basic FGF, and FGF-5 were only slightly induced (2- to 10-fold) during wound healing, and there was no expression of FGF-3, FGF-4, and FGF-6 detected in normal and wounded skin. High levels of FGF receptor 1 and FGF receptor 2 mRNA and low levels of FGF receptor 3 mRNA were found in both normal and wounded skin. No change in the levels of these transcripts was detected during wound healing. In situ hybridization studies revealed highest levels of KGF mRNA expression in the dermis at the wound edge and in the hypodermis below the wound. In contrast, mRNA encoding the receptor of this growth factor (a splice variant of FGF receptor 2) was predominantly expressed in the epidermis. These results suggest that basal keratinocytes are stimulated by dermally derived KGF during wound healing and implicate a unique role of this member of the FGF family in wound repair.

Animals↗
PubMed1992

Secreted inhibitors of metalloproteinases (IMPs) that are distinct from TIMP.

The tissue inhibitor of metalloproteinases (TIMP, M(r) 30,000) is secreted by many cell and tissue types and has been shown to inhibit most secreted mammalian metalloproteinases. In matrix and tissue invasion assays, the inactivation or removal of TIMP enhances invasiveness. However, many of the cells that secrete TIMP also secrete other metalloproteinase inhibitors. By analysis of medium conditioned by various endothelial, mesenchymal, and neural cells on SDS-.substrate-polyacrylamide-inhibitor gels (reverse zymograms), we have detected at least three other distinct inhibitors of metalloproteinases (IMPs). Some or all of these IMPs have been detected in secretions of mouse, rabbit, sheep, and human cells and are all smaller in apparent molecular size than TIMP (IMP-1, M(r) 26,000; IMP-2, M(r) 21,000; IMP-3, M(r) 18,000). These IMPs are not proteolytic degradation products of TIMP nor do they represent nonglycosylated TIMP. The IMPs do not cross-react in the native or denatured state with any of several anti-TIMP antibodies. The IMPs appear to be regulated independently of each other and of TIMP. In vitro, the complex consisting of one of the IMPs, or TIMP, and a metalloproteinase can be dissociated into functional inhibitor and metalloproteinase. Whether this characteristic is significant in vivo is not known. IMP-2 has been purified from several sources and shares sequence homology with TIMP, suggesting that the IMPs and TIMP may constitute a gene family. The most significant characteristic of IMP-2 is that it appears to preferentially inhibit, on a mole:mole basis, the M(r) 68,000 gelatinase rather than collagenase or stromelysin.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1991

Binding of tissue inhibitor of metalloproteinases 2 to two distinct sites on human 72-kDa gelatinase. Identification of a stabilization site.

We have identified a binding site for tissue inhibitors of metalloproteinases 2 (TIMP-2) on human 72-kDa gelatinase that is distinct from the active site. 72-kDa progelatinase is found in a complex with TIMP-2 in the medium of cultured cells and can be activated with organomercurial compounds to yield a gelatinolytic proteinase that remains bound to TIMP-2. Removal of TIMP-2 from 72-kDa progelatinase by reverse-phase high performance liquid chromatography, followed by reconstitution of the progelatinase in neutral pH buffer, results in autocatalytic activation. When samples of autoactivated gelatinase were blotted onto nitrocellulose, then probed with 125I-TIMP-2, we found a 29-kDa peptide that was capable of binding TIMP-2. We isolated this fragment and identified it as the region of gelatinase from amino acid 414 to the carboxyl terminus in the primary amino acid sequence of progelatinase. This portion of the molecule does not contain the putative zinc- or gelatin-binding sites and is proteolytically inactive. Incubation of 125I-TIMP-2 with 72-kDa progelatinase-TIMP-2 complexes resulted in a concentration-dependent exchange of labeled TIMP-2 with unlabeled TIMP-2, in both the presence and absence of the metalloproteinase inhibitor 1,10-phenanthroline. Saturation binding kinetics for the active site of 72-kDa gelatinase were measured in pools of the 43-kDa active fragment that results from the autoactivation of 72-kDa progelatinase; this fragment has no carboxyl-terminal TIMP-2 binding capability. Binding of 125I-TIMP-2 to the active site was completely inhibited by 1,10-phenanthroline. Binding kinetics for the putative stabilization site were determined with isolated 72-kDa progelatinase. In the presence of 1,10-phenanthroline, 72-kDa progelatinase bound 125I-TIMP-2 but not 125I-TIMP-1. Scatchard analysis yielded an approximate dissociation constant (Kd) of 0.72 nM for the active site and 0.42 nM for the stabilization site.

Autoradiography↗
PubMed1991

Preferential inhibition of 72- and 92-kDa gelatinases by tissue inhibitor of metalloproteinases-2.

Transformed human fibroblasts secrete two structurally and functionally related inhibitors of matrix metalloproteinases, tissue inhibitor of metalloproteinases (TIMP) 1 and 2. In assays measuring the relative inhibitory capability of TIMP-1 and TIMP-2 against autoactivated 72-kDa gelatinase, which consists of two major active peptides and several inactive fragments, TIMP-2 was more effective than TIMP-1. The isolated 42.5-kDa active fragment that formed as a result of the autoactivation of 72-kDa gelatinase showed the greatest preference for TIMP-2; at half-maximal inhibition, TIMP-2 was greater than 10-fold more effective than TIMP-1. TIMP-2 was also greater than 2-fold more effective than TIMP-1 at inhibiting 72-kDa gelatinase-TIMP-2 complexes activated with 4-aminophenylmercuric acetate, and greater than 7-fold more effective than TIMP-1 at inhibiting 92-kDa gelatinase activated with 4-aminophenylmercuric acetate. Furthermore, these active gelatinases preferentially bound 125I-TIMP-2 when incubated with equal amounts of radiolabeled TIMP-1 and TIMP-2. The ratios of 125I-TIMP-2/125I-TIMP-1 binding to 92-kDa gelatinase, autoactivated 72-kDa gelatinase, and 42.5-kDa fragment were 4.4, 10, and 33, respectively. On the other hand, interstitial collagenase was inhibited by TIMP-1 greater than 2-fold more effectively than TIMP-2 in assays measuring cleavage of loose collagen fibrils.

Cell Line↗
PubMed1991

Regulation of the autoactivation of human 72-kDa progelatinase by tissue inhibitor of metalloproteinases-2.

To study the activation of human 72-kDa gelatinase, and its relation to tissue inhibitor of metalloproteinases 2 (TIMP-2), we purified human 72-kDa progelatinase both as a complex with TIMP-2 and as a free proteinase. Activation of progelatinase-TIMP-2 complexes with 4-aminophenylmercuric acetate yielded gelatinolytically active enzyme migrating at 62 kDa. TIMP-2 remained bound to the active enzyme. Removal of TIMP-2 from progelatinase by reverse-phase high performance liquid chromatography in the presence of trifluoroacetic acid, followed by complete dialysis in neutral pH buffer, resulted in multiple fragments. These fragments were formed as a result of the cleavage of 72-kDa progelatinase at several locations. Cleavage at the amino terminus was restricted to the removal of the propeptide, except in the case of degradation leading to inactive fragments. Two active species autocatalytically evolved upon removal of TIMP-2 from progelatinase. The 62 kDa-activated gelatinase lacked the amino-terminal propeptide, which is known to be removed upon treatment with 4-aminophenylmercuric acetate. In addition, an active 42.5-kDa fragment lacking both the propeptide and a portion of the carboxyl terminus was formed. This low-molecular-weight active form of 72-kDa progelatinase retained its ability to bind and degrade gelatin. Self-activation and degradation of 72-kDa progelatinase can be prevented by agents that inhibit metalloproteinases, including 1,10-phenanthroline. Evidence presented here suggests that TIMP-2 binds to a stabilization site that is independent of the active site. This stabilization site does not bind TIMP-1 (TIMP). Occupation of this site by TIMP-2 prevents autocatalytic activation and degradation but does not prevent gelatinolysis by the enzyme-inhibitor complex.

Ataxia Telangiectasia↗
PubMed1990

Expression of metalloproteinases and metalloproteinase inhibitors by fetal astrocytes and glioma cells.

Metalloproteinases have been implicated as important factors mediating the tissue migration of a variety of normal and transformed cells. The conditioned medium (CM) of fetal human astrocytes and five glioma cell lines did not degrade azocoll in suspension, but several proteolytic activities, inhibitable by 1,10-phenanthroline, were detected on sodium dodecyl sulfate-polyacrylamide gels containing gelatin. Both cell types secreted three major proteolytic species (Mr 65,000, 57,000, and 52,000). Two of the glioma lines secreted an additional proteinase (Mr 92,000). After treatment with 12-O-tetradecanoylphorbol-13-acetate, the secretion of the Mr 92,000, 57,000, and 52,000 proteinases was induced or enhanced in all of the cells. The Mr 92,000 and 65,000 proteinases bound specifically to a gelatin affinity column. When purified by preparative gel electrophoresis, the Mr 65,000 proteinase was found to degrade type IV procollagen. The Mr 57,000 and 52,000 species were precipitated by anticollagenase IgG. Tissue inhibitor of metalloproteinases was detected in the CM of all of the cells by substrate gel analysis and immunoprecipitation of [35S]methionine-labeled proteins with anti-tissue inhibitor of metalloproteinases IgG. The glioma lines also secreted various amounts of two smaller inhibitors of metalloproteinases (IMPs), also seen in rabbit brain capillary endothelial cell CM (IMP-1 at Mr 22,000 and IMP-2 at Mr 19,000), and an inhibitor not previously identified (IMP-3 at Mr 16,500). 12-O-Tetradecanoylphorbol-13-acetate stimulated the secretion of tissue inhibitor of metalloproteinases in all of the cells and induced IMPs in some of the glioma lines. When gel filtration chromatography of concentrated CM was used to resolve inhibitors from proteinases, the isolated proteinases had activity against azocoll and the glycoprotein and collagen components of an in vitro model of the extracellular matrix. The secretion of a battery of metalloproteinases by astrocytes may be important in facilitating astrocytic migration during development and in pathological conditions such as inflammation or local invasion of astrocytic neoplasms.

Astrocytes↗
PubMed1988

Wound macrophages express TGF-alpha and other growth factors in vivo: analysis by mRNA phenotyping.

The presence of macrophages is required for the regeneration of many cell types during wound healing. Macrophages have been reported to express a wide range of mitogenic factors and cytokines, but none of these factors has been shown in vivo to sustain all the wound-healing processes. It has been suggested that transforming growth factor-alpha (TGF-alpha) may mediate angiogenesis, epidermal regrowth, and formation of granulation tissue in vivo. Macrophages isolated from a wound site, and not exposed to cell culture conditions, expressed messenger RNA transcripts for TGF-alpha, TGF-beta, platelet-derived growth factor A-chain, and insulin-like growth factor-1. The expression of these transcripts was determined by a novel method for RNA analysis in which low numbers of mouse macrophages were isolated from wound cylinders, their RNA was purified and reverse-transcribed, and the complementary DNA was amplified in a polymerase chain reaction primed with growth factor sequence-specific primers. This single-cell RNA phenotyping procedure is rapid and has the potential for quantification, and mRNA transcripts from a single cell or a few cells can be unambiguously demonstrated, with the simultaneous analysis of several mRNA species. Macrophages from wounds expressed TGF-alpha antigen, and wound fluids contained TGF-alpha. Elicited macrophages in culture also expressed TGF-alpha transcripts and polypeptide in a time-dependent manner after stimulation with modified low-density lipoproteins and lipopolysaccharide endotoxin, which are characteristic of the activators found in injured tissues.

Animals↗
PubMed1988

The inhibitory complex of human alpha 1-proteinase inhibitor and human leukocyte elastase is a neutrophil chemoattractant.

An inhibitor-proteinase complex consisting of human alpha 1-PI and human leukocyte elastase is chemotactic for human neutrophils. The chemotactic activity is optimal at 1 nM and is associated only with the alpha 1-PI portion of the complex. Neither HLE in the complex, free HLE, nor native alpha 1-PI possesses chemotactic activity for human neutrophils. alpha 1-PI in complex is hydrolyzed at the Met-358-Ser-359 bond. The chemotactic activity is associated with the Mr 4,200 fragment of alpha 1-PI that has Ser-359 as its NH2 terminus. The region of the HLE-alpha 1-PI complex that stimulates chemotaxis appears to be the same as that of the Mr 4,200 fragment generated by hydrolysis of the Pro-357-Met-358 bond during proteolytic inactivation of alpha 1-PI. The data suggest the presence of a neutrophil surface receptor bound by alpha 1-PI after the formation of a complex with HLE or after proteolytic degradation. This receptor may play a role in clearance of these modified alpha 1-PI molecules.

Blood Proteins↗
PubMed1988

Alpha 1-proteinase inhibitor is a neutrophil chemoattractant after proteolytic inactivation by macrophage elastase.

Mouse macrophage elastase, a metalloproteinase, catalytically inactivates human alpha 1-proteinase inhibitor (alpha 1-PI) by attacking a single peptide bond between Pro357 and Met358, resulting in Mr = 4,200 and 47,800 fragments. We show here that this proteolytically inactivated alpha 1-PI is a potent chemotactic factor for human neutrophils at a concentration of 1 nM. The chemotactic response is equivalent to that elicited by formyl-methionyl-leucyl-phenylalanine. Native alpha 1-PI does not stimulate chemotaxis. Purification of the two fragments of alpha 1-PI that result from proteolysis by macrophage elastase indicated that the Mr = 4,200 fragment is responsible for the chemotactic activity. However, the two proteolysis fragments do not dissociate from each other under physiologic conditions. Therefore, the ability of proteolytically inactivated alpha 1-PI to act as a mediator of inflammation is due to rearrangement of the alpha 1-PI molecule rather than to release of a cleavage fragment.

Animals↗
PubMed1987

Interaction of mouse macrophage elastase with native and oxidized human alpha 1-proteinase inhibitor.

Native and oxidized alpha 1-proteinase inhibitor (alpha 1-PI) were compared as substrates for the metalloproteinase macrophage elastase. At substrate concentrations at which native alpha 1-PI was readily degraded by macrophage elastase, oxidized alpha 1-PI was hardly degraded at all. Incubation of macrophage elastase with oxidized alpha 1-PI before the addition of native alpha 1-PI showed that oxidized alpha 1-PI was not an inhibitor of macrophage elastase. Competition experiments with up to twofold excess oxidized alpha 1-PI did not interfere with the degradation of native alpha 1-PI by macrophage elastase. Sequence analysis of amino acids in degraded native alpha 1-PI showed that macrophage elastase attacked a single peptide bond between Pro-357 and Met-358, the latter representing the P1 reactive-site residue of alpha 1-PI. In oxidized alpha 1-PI, Met-358 was converted to methionine sulfoxide and macrophage elastase hydrolyzed the bond between Phe-352 and Leu-353. These data suggest that methionine may be the primary cleavage site for macrophage elastase and not leucine, as previously thought.

Amino Acid Sequence↗
PubMed1986

Secretion of metalloproteinases by stimulated capillary endothelial cells. I. Production of procollagenase and prostromelysin exceeds expression of proteolytic activity.

We have characterized the biosynthesis of two metalloproteinases, procollagenase and prostromelysin, by rabbit brain capillary endothelial cells (RBCE) by means of immunochemical, biosynthetic, and functional assays. Unstimulated RBCE secreted no detectable metalloproteinases. Secretion of both procollagenase and prostromelysin was induced within 6 h by treating the cells with 50 ng/ml 12-O-tetradecanoylphorbol-13-acetate. In treated cells, the two proenzymes accounted for up to 20% of the [35S]methionine-labeled secreted proteins; about 15 micrograms of each protein was secreted in 48 h by 10(6) RBCE. Although RBCE secreted approximately as much procollagenase and prostromelysin as did rabbit fibroblasts, virtually no enzyme activity could be measured in RBCE-conditioned medium, even after activation of the proenzymes by trypsin or an organomercurial agent.

Animals↗
PubMed1986

Secretion of metalloproteinases by stimulated capillary endothelial cells. II. Expression of collagenase and stromelysin activities is regulated by endogenous inhibitors.

Rabbit brain capillary endothelial cells treated with 12-O-tetradecanoylphorbol-13-acetate produce the metalloproteinases, procollagenase and prostromelysin, as up to 20% of their total secreted protein. However, little or no catalytic activity of these enzymes can be found after treatment with either trypsin or an organomercurial agent, which are able to activate the proenzymes in the medium from stimulated rabbit fibroblasts. We now have shown that enzyme activities of procollagenase and prostromelysin are revealed after conditioned medium is analyzed by gel filtration chromatography or by electrophoresis on sodium dodecyl sulfate-substrate gels. In both systems, the metalloproteinases were separated from metalloproteinase inhibitors. The major inhibitor of Mr = 30,000 from capillary endothelial cells was immunologically identical with the rabbit tissue inhibitor of metalloproteinases. Two additional inhibitors of metalloproteinases at Mr = 22,000 and 19,000 were also observed. Inhibitors were present in the conditioned medium from rabbit fibroblasts in much lower quantities and were also qualitatively different. When gel filtration chromatography was used to remove the tissue inhibitor of metalloproteinases from medium conditioned by stimulated capillary endothelial cells, both activatable procollagenase and prostromelysin were readily demonstrable. These data suggest that endogenous inhibitors regulate the expression of metalloproteinases secreted by endothelial cells.

Animals↗
PubMed1986

Some protease inhibitors are also inhibitors of poly(ADP-ribose) polymerase.

The low-molecular-weight peptide protease inhibitors, tosyl-lysine-chloromethyl ketone, antipain and leupeptin, inhibited poly(ADP-ribose) [poly(ADP-Rib)] polymerase in permeable cells. The concentrations required for 50% inhibition were 3.6, 5 and 29 mM, respectively. Two peptides without protease inhibitor activity, fibrinopeptide A and phenylalanine-leucine-(glutamine)2-leucine, also inhibited poly (ADP-Rib) synthesis; doses required for 50% inhibition were 0.37 and 11.2 mM, respectively. These concentrations lie within a range bracketed by the 50% inhibition concentrations of the strong and weak poly(ADP-Rib) synthesis inhibitors, 3-amino-benzamide (0.15 mM) and caffeine (greater than 100 mM), respectively. N-Ethylmaleimide also inhibited poly(ADP-Rib) synthesis, at a 50% inhibitory dose of 0.3 mM, in the absence of exogenous thiol reagents. High-molecular-weight protease inhibitors, such as soybean (including Bowman-Birk reagent) and lima bean trypsin inhibitors and human alpha 1-protease inhibitor, had no effect on poly(ADP-Rib) synthesis up to 2 mg/ml. Interference with transformation and other cellular effects that have been reported in carcinogen-damaged cells treated with low-molecular-weight peptide protease inhibitors may therefore involve common mechanisms with poly(ADP-Rib) inhibitors. Similar effects of high-molecular-weight protease inhibitors presumably involve different mechanisms.

Antipain↗
PubMed1986

Effect of lactate, pyruvate, and pH on secretion of angiogenesis and mitogenesis factors by macrophages.

Macrophages secrete substances that stimulate angiogenesis and mitogenesis at wound sites. To test whether metabolic characteristics of the wound such as high lactate concentration and low pH may regulate the expression of these substances, macrophages were cultured in lactalbumin hydrolysate and then in increasing concentrations of lactate, pyruvate, and hydrogen ions, and cell-free supernatants were collected. Cultures incubated in concentrations of lactate approximating those observed in wounds secreted an angiogenesis factor or factors, but cultures incubated in equivalent concentrations of pyruvate or at pH 6.2 did not. These observations seem to be specific to macrophages because capillary endothelial cells and fibroblasts failed to secrete an angiogenesis factor when cultured under hypoxic conditions or in high concentrations of lactate. None of these conditions changed the expression of macrophage mitogens.

Angiogenesis Inducing Agents↗
PubMed1985

Cell interactions in post-traumatic fibrosis.

Models of reparative fibrosis, or wound healing, disclose a basic spatial relationship between inflammatory cells, fibroblasts, dividing fibroblasts and angiogenic endothelial cells. Many components of the extracellular fluid, especially steep gradients of PO2, PCO2, pH, glucose and lactate, have been measured. After components of coagulation play out their role in the first few days after injury, macrophages sense excitatory substances and signs of respiratory distress, and secrete chemoattractants for endothelial cells and fibroblasts. If excitatory substances (such as silica) are eliminated, active fibrosis ends with the restoration of the energy supply. Some of the mitogens and chemoattractants acting as intercellular messengers between these cells have been isolated and identified.

Animals↗