Biomedical subjects

R Poorman

Publications and source records attributed to R Poorman.

10 recordsLinked to original sources

PubMed1995

A heme-binding protein from hemolymph and oocytes of the blood-sucking insect, Rhodnius prolixus. Isolation and characterization.

A heme-binding protein has been isolated and characterized from both the hemolymph and oocytes of the blood-sucking insect, Rhodnius prolixus. The protein from both sources is identical in most aspects studied. The Rhodnius heme-binding protein (RHBP) is composed of a single 15-kDa polypeptide chain coiled in a highly alpha-helical structure which binds non-covalently one heme/polypeptide chain. This RHBP is not produced by limited degradation of hemoglobin from the vertebrate host, since specific polyclonal antibodies against it do not cross-react with rabbit hemoglobin, and since it differs from hemoglobin in having a distinct amino-acid composition and NH2-terminal sequence. The spectrum of the dithionite-reduced protein has peaks at 426, 530, and 559 nm and resembles that of a b-type cytochrome. RHBP from hemolymph is not saturated with heme and promptly binds heme added to the solution. The oocyte protein, on the other hand, is fully saturated and is not capable of binding additional heme.

Amino Acid Sequence↗

PubMed1995

The tetracycline analogs minocycline and doxycycline inhibit angiogenesis in vitro by a non-metalloproteinase-dependent mechanism.

The tetracycline analogs minocycline and doxycycline are inhibitors of metalloproteinases (MMPs) and have been shown to inhibit angiogenesis in vivo. To further study the mechanism of action of these compounds we tested them in an in vitro model of angiogenesis: aortic sprouting in fibrin gels. Angiogenesis was quantitated in this system by a unique application of planar morphometry. Both compounds were found to potently inhibit angiogenesis in this model. To further characterize the activity of these compounds against MMPs, we determined the IC50S of both compounds against representatives of three classes of metalloproteinases: fibroblast collagenase, stromelysin, and gelatinase A. Doxycycline was found to inhibit collagenase, gelatinase A and stromelysin with IC50S of 452 microM, 56 microM and 32 microM, respectively. Minocycline was found to inhibit only stromelysin in the micromolar range with an IC50 of 290 microM. Since these results suggest that these compounds may not have been inhibiting in vitro angiogenesis by an MMP-dependent mechanism, we decided to test the effects of the potent MMP inhibitor BB-94. This compound failed to inhibit aortic sprouting in fibrin gels, thus strongly suggesting that both doxycycline and minocycline act by an MMP-independent mechanism. These results have implications for the mechanism of action of tetracycline analogs, particularly where they are being considered for the treatment of disorders of extracellular matrix degradation including periodontal disease, arthritis, and tumor angiogenesis.

Animals↗

PubMed1994

Interstitial collagenase is required for angiogenesis in vitro.

Human umbilical vein endothelial cells (HUVECs) invade collagen gels and establish vascular-like structures within the gel following stimulation with phorbol esters. This process was quantitated by measuring release of radioactivity from gels composed of [3H]collagen. Collagen was steadily degraded over the period of several weeks by phorbol ester-treated cells while little collagenolysis by cells not receiving phorbol ester was noted. Examination of matrix metalloproteinases (MMPs) secreted by HUVECs revealed a prominent induction of interstitial collagenase. Production of the mature forms of gelatinase A was also stimulated, as was the secretion of gelatinase B. Stromelysin was not detected. Two inhibitors of MMPs, the naturally occurring tissue inhibitor of metalloproteinases (TIMP; 10 micrograms/ml) and the synthetic, peptide inhibitor BB-94 (1 microM) were both effective at blocking HUVEC-mediated collagen degradation. Morphological examination of control, PMA-treated HUVECs, as well as PMA-treated HUVECs receiving TIMP or BB-94, revealed that MMP inhibition resulted in a block to invasion and tubule formation within the collagen gels. Similar results for MMP expression and inhibition of tubule formation in vitro were obtained with human dermal microvascular endothelial cells. Examination of collagen proteolytic fragments revealed that both BB-94 and TIMP blocked cleavage of the alpha 1 and alpha 2 chains of type I collagen and the appearance of tropocollagen fragments A and B, demonstrating that the inhibitors were acting directly upon interstitial collagenase. Our results demonstrate that interstitial collagenase is required for angiogenesis in vitro.

Amino Acid Sequence↗

PubMed1993

Inhibition of acetaminophen and lorazepam glucuronidation in vitro by probenecid.

The effect of probenecid on glucuronidation of acetaminophen and lorazepam in hepatic microsomes from various species was studied to see if in vitro results were consistent with previous in vivo observations. Mouse, rat, and human microsomes were incubated with acetaminophen and probenecid while monkey microsomes were incubated with lorazepam and probenecid. Glucuronidation rates in all species varied with substrate, protein, and detergent concentrations. Mice exhibited faster rates of glucuronidation than rats or humans. All species showed inhibition of glucuronidation of acetaminophen or lorazepam when probenecid was added. Analysis suggested competitive inhibition. Thus, in vitro studies support in vivo results and confirm that the inhibition takes place at the hepatic level.

Acetaminophen↗

PubMed1992

Probing the structure-function relationship of nerve growth factor.

We compared the receptor binding, antigenicity, biological activation, and cell-mediated proteolytic degradation properties of mouse nerve growth factor (mNGF) and human NGF (hNGF). The affinity of hNGF toward human NGF-receptor is greater than that of mNGF, but the affinity of mNGF toward rat NGF-receptor is greater than that of hNGF. Thus, the specificity of the interaction between NGF and its receptor resides both on the NGF and on its receptor. Using a group of anti-NGF monoclonal antibodies that competitively inhibit the binding of NGF to receptor, sites differing between mNGF and hNGF were detected. Together, these results indicate that the sites on hNGF and mNGF, responsible for binding to NGF-receptor, are similar but not identical. In comparing the relative abilities of mNGF and hNGF to stimulate a biological response in PC12 cells, we observed that mNGF was better at stimulating neurite outgrowth than was hNGF, consistent with the differences observed for receptor binding affinity. However, the ED50 for biological activation is approximately 100-fold lower than the Kd for receptor occupancy, and, thus, the dose-response curve is not consistent with a simple activation proportional to receptor occupancy. The data are consistent with a model requiring a low-level threshold occupancy of NGF-receptor (Kd = 10(-9) M) in order to stimulate full biological activity. Finally, we observed the degradation of NGF by PC12 cells. We found that the NGF molecule is significantly degraded via a receptor-mediated uptake mechanism. Together, the data provide insight into regions of the NGF molecule involved in contacts with the receptor leading to formation of the NGF:NGF-receptor complex. Additionally, they establish the link between occupancy of receptor and biological activation and the requirement for receptor-mediated uptake in order to degrade NGF proteolytically in cultured PC12 cells.

Animals↗

PubMed1991

Single-step purification and biological activity of human nerve growth factor produced from insect cells.

Human nerve growth factor (NGF) was cloned and engineered for expression in a baculovirus-infected Spodoptera frugiperda (SF-9) insect cell system. Culture supernatants contained 2-3 mg/L of recombinant human NGF. The human NGF produced by this system was purified to apparent homogeneity with a single-step affinity chromatography procedure using a high-affinity monoclonal antibody originally raised against murine NGF. The purification procedure yielded 1-2 mg of pure, human NGF per liter of culture supernatant; i.e., approximately 60% recovery of the human NGF originally released into the culture medium. Although the gene transfected into the SF-9 cells coded for pro-NGF, the NGF recovered after purification was greater than 95% fully processed, mature protein. The KD for the affinity of the pure, recombinant human NGF for NGF receptor in PC12 membranes is 0.20 +/- 0.05 nM. Activation of neurite outgrowth in PC12 cells occurs with ED50 values of 85 +/- 20 pM and 9.6 +/- 1.5 pM for a 3-day primary response and a 1-day secondary response, respectively. The pure, recombinant human NGF also stimulates a significant increase in dopamine content of PC12 cells with an ED50 of 5.8 +/- 2.7 pM. These binding and biological activation properties are consistent with values observed using murine NGF purified from submaxillary glands.

Amino Acid Sequence↗

PubMed1990

Purification and characterization of acyl carrier protein from two cyanobacteria species.

The acyl carrier protein (ACP), an essential protein cofactor for fatty acid synthesis, has been isolated from two cyanobacteria: the filamentous, heterocystous, Anabaena variabilis (ATCC 29211) and the unicellular Synechocystis 6803 (ATCC 27184). Both ACPs have been purified to homogeneity utilizing a three-column procedure. Synechocystis 6803 ACP was purified 1800-fold with 67% yield, while A. variabilis ACP was purified 1040-fold with 50% yield. Yields of 13.0 micrograms ACP/g Synechocystis 6803 and 9.0 micrograms ACP/g A. variabilis were achieved. Amino acid analysis indicated that these ACPs were highly charged acidic proteins similar to other known ACPs. Sequence analysis revealed that both cyanobacterial ACPs were highly conserved with both spinach and Escherichia coli ACP at the phosphopantetheine prosthetic group region. Examining the probability of alpha-helix and beta-turn regions in various ACPs, showed that cyanobacterial ACPs were more closely related to E. coli ACP than spinach ACP I. Immunoblot analysis and a competitive binding assay for ACP illustrated that both ACPs bound poorly to spinach ACP I antibody. SDS/PAGE and native PAGE of Synechocystis 6803 ACP and A. variabilis ACP showed that cyanobacteria ACPs co-migrated with E. coli ACP and had relative molecular masses of 18,100 and 17,900 respectively. Both native and urea gel analysis of acyl-ACP products from fatty acid synthase reactions demonstrated that bacterial ACPs and plant ACP gave essentially the same metabolic products when assayed using either bacterial or plant fatty acid synthase. A. variabilis and Synechocystis 6803 ACP could be acylated using E. coli acyl ACP synthetase.

Acyl Carrier Protein↗

PubMed1989

An experimental method for the determination of enzyme-competitive inhibitor dissociation constants from displacement curves: application to human renin using fluorescence energy transfer to a synthetic dansylated inhibitor peptide.

We developed a facile procedure for the determination of enzyme-competitive inhibitor dissociation constants over a wide range of potencies at any ratios of enzyme, labeled ligand, and inhibitor. The assay uses displacement curves and a fluorescent-labeled ligand to allow experimental determination of dissociation constants (Kd's) of inhibitors of human renin, a highly specific enzyme, for which numerous high affinity (up to 100 pM) inhibitors have been synthesized. The procedure involves binding a dansylated competitive inhibitor, U80215, followed by its displacement by an unlabeled inhibitor of renin. Binding of U80215 is monitored by fluorescent energy transfer from the renin tryptophans to the dansyl moiety; displacement of U80215 by an unlabeled inhibitor is monitored by a reversal of this process. The procedures may be used to determine the potencies of unlabeled inhibitors up to 100 pM affinities and to determine kinetic binding constants. The concepts described should also be useful in other protein/ligand systems.

Amino Acid Sequence↗

PubMed1987

Inaccessibility of tryptophan residues of recombinant human renin to quenching agents.

The fluorescence quenching of the three tryptophan residues of recombinant human renin was determined using ionic and penetrating quenchers. Tryptophans 44,200, and 312 of recombinant human renin were found to be totally inaccessible to the ionic quenchers cesium and iodide and only partially accessible to the penetrating quencher acrylamide. The renin had a fluorescence emission maximum at 325 nm which was made up of three separate components as determined by second derivative spectroscopy. These data are in accord with solvent accessibility calculations from three-dimensional models of human renin but differ from findings published previously from similar analysis of mouse submandibular gland renin (Quay, S. C., Heropoulous, A., Commes, K., Dzau, V. J. (1985) J. Biol. Chem. 260, 15055-15058), which is 68% identical in sequence to human renin.

Acrylamide↗

PubMed1979

Inhibition of carboxypeptidase A catalyzed peptide hydrolysis by 3-phenylpropanoate at activating and nonactivating substrate concentrations.

The carboxypeptidase A catalyzed hydrolyses of five structurally related dipeptide substrates in the presence of the inhibitor 3-phenylpropanoate have been studied. At nonactivating substrate concentrations, 3-phenylpropanoate is a mixed inhibitor of carbobenzoxyglycyl-L-phenylalanine hydrolysis and a noncompetitive inhibitor of the hydrolyses of benzoylglycyl-L-phenylalanine, cinnamoylglycyl-L-phenylalanine, hydrocinnamoylglycyl-L-phenylalanine, and acetylglycyl-L-phenylalanine. When carbobenzoxyglycyl-L-phenylalanine and benzoylglycyl-L-phenylalanine exhibit substrate activation, inhibition by 3-phenylpropanoate is mixed but appears to be mostly competitive. Proposed here is a site for the binding of 3-phenylpropanoate along with a kinetic mechanism consistent with these data.

Carboxypeptidases↗