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Biomedical subjects

D A Lawrence

Publications and source records attributed to D A Lawrence.

At least 109 records · Page 6Linked to original sources
PubMed1995

The effects of interleukin-1 beta and tumor necrosis factor-alpha on in vitro colony formation by human hematopoietic progenitor cells exposed to doxorubicin or hydroquinone.

Previous studies have shown that treatment of bone marrow (BM) cells with interleukin-1 beta (IL-1 beta) or tumor necrosis factor-alpha (TNF-alpha) can protect hematopoietic progenitor cells (HPC) from the toxic effects of 4-hydroperoxycyclophosphamide (4HC) or gamma-irradiation. Since doxorubicin (DX) and hydroquinone (HQ) may inhibit hematopoiesis through mechanisms similar to 4HC and gamma-irradiation, it was of interest to determine whether IL-1 beta or TNF-alpha could protect HPC from DX and HQ as well. Bone marrow mononuclear cells (BMMNC) or purified HPC (pHPC) were exposed to 50 ng/mL IL-1 beta or 25 ng/mL TNF-alpha alone or in combination with DX or HQ for 22 hours at physiological O2 partial pressure and temperature. The cells were washed free of the cytokines and toxicants and plated in cytokine-containing semisolid medium. Under these concurrent cytokine +/- toxicant treatment conditions, neither IL-1 beta nor TNF-alpha significantly affected progenitor cell frequencies (assessed as CFU-C) or lineage commitment compared with the medium-treated controls. Treatment with either 100 nM DX or 30 microM HQ, however, reduced CFU-C frequencies by approximately 70%. When BMMNC were used, treatment with neither IL-1 beta nor TNF-alpha consistently protected CFU-C from either DX or HQ. In contrast, using pHPC, IL-1 beta or TNF-alpha treatment conferred nearly two-fold protection of CFU-C from DX in all donors tested. TNF-alpha protected CFU-C from HQ using pHPC from all but one donor, while IL-1 beta did not protect CFU-C from HQ. Using phPC, maximum protection of CFU-C from DX was reached at IL-1 beta or TNF-alpha concentrations above 10 ng/mL or 1 ng/mL, respectively. Treatment of pHPC with TNF-alpha for at least 8 hours was necessary before significant protection from DX could be detected. Therefore, we conclude that IL-1 beta and TNF-alpha can act directly on human HPC to protect them from the inhibitory effects of DX and that, to a lesser extent, TNF-alpha can directly protect HPC from HQ.

Adolescent↗
PubMed1995

The spatial coding strategies of one-year-old infants in a locomotor search task.

The ability of 1-year-old infants to remember the location of a nonvisible target was investigated in 3 experiments. Infants searched for a toy hidden in one of many possible locations within a circular bounded space. The presence, number, and spatial arrangement of local cues or "landmarks" within this space were varied. The results of Experiment 1 showed that search performance was highly successful when a landmark was coincident with the location of the toy ("direct"), but less successful when a landmark was adjacent to the target location ("indirect"). The results of Experiment 2 suggested that search with an indirect landmark may be more fragile than search with no landmarks at all. In Experiments 3a and 3b, 2 different configurations of indirect landmarks were employed; search performance was equally poor with both of these and was inferior to search with no landmarks. It is concluded that infants of this age are able to associate a nonvisible target with a direct landmark and are able to code the distance and direction of a target with respect to themselves or with respect to the larger framework. However, there was no evidence that they can code the distance and direction of a target relative to another object. The difficulty of coding with indirect landmarks is interpreted in terms of cognitive complexity and conflict between spatial strategies.

Child Development↗
PubMed1994

Serpin reactive center loop mobility is required for inhibitor function but not for enzyme recognition.

One feature that distinguishes all of the inhibitory members of the serpin gene family is the presence of a small uncharged residue at the P14 position of the reactive center loop. In this report we examine the effects of mutations at this position, in the serpin, plasminogen activator inhibitor type 1 (PAI-1). Replacement of the native P14 Thr-333 residue by an Arg (Thr-333-->Arg) resulted in complete loss of inhibitory activity toward tissue-type plasminogen activator and urokinase-type plasminogen activator. Comparison of the binding of the mutant inhibitor and wild type PAI-1 (WTPAI-1) to anhydrotrypsin indicated that the initial interaction of the two inhibitors with proteases was identical. However, whereas WTPAI-1 forms SDS-stable complexes with both plasminogen activators, the mutant PAI-1 was efficiently cleaved as a substrate. Amino-terminal sequence analysis indicated that cleavage of the mutant PAI-1 occurred at its reactive center P1-P1' Arg-Met bond. Thermal denaturation studies of native and cleaved PAIs indicated that native Thr-333-->Arg mutant had a thermal stability identical to active WTPAI-1 and that both proteins became significantly more stable following cleavage by elastase (cleaved at the P4-P3 bond). Finally, the function of recombinant PAI-1 variants containing 15 of the possible 19 amino acid substitutions at P14 were analyzed. While residue size appeared to have little effect on inhibitory activity, the presence of either a positive or a negative charge at P14, converted PAI-1 to a substrate. Taken together, these results suggest that while insertion of the reactive center loop is not essential for protease binding, it is a necessary second step required for inhibitor function. The presence of a charged residue at P14 can retard this insertion, resulting in conversion of the serpin to a substrate.

Base Sequence↗
PubMed1994

Regions involved in binding of urokinase-type-1 inhibitor complex and pro-urokinase to the endocytic alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein. Evidence that the urokinase receptor protects pro-urokinase against binding to the endocytic receptor.

The alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein (alpha 2MR/LRP) binds several ligands, including complex between the two chain urokinase-type plasminogen activator (uPA) and type-1 plasminogen activator inhibitor (PAI-1), and the single chain zymogen pro-urokinase (pro-uPA). We have used truncated variants of uPA and PAI-1 as well as Fab fragments of monoclonal antibodies with known epitopes to identify regions in the uPA.PAI-1 complex and in pro-uPA involved in binding to alpha 2MR/LRP.uPA.PAI-1 complex bound with high affinity (EC50 about 0.4 nM) via contacts in the PAI-1 moiety as well as the uPA serine proteinase domain and the uPAA chain. Pro-uPA bound with lower affinity (EC50 about 10 nM), and efficient binding to alpha 2MR/LRP was dependent on contact with both the A chain and the serine proteinase domain. We analyzed the effect of complex formation with the urokinase receptor since this is the primary target for binding of uPA.PAI-1 and pro-uPA at the cell surface, and since it has been demonstrated that urokinase receptor-bound uPA.PAI-1 complex is internalized following interaction with alpha 2 MR/LRP (Nykjaer, A., Petersen, C. M., Møller, B., Jensen, P.H., Moestrup, S.K., Holtet, T.L., Etzerodt M., Thøgersen, H.C., Munch, M., Andreasen, P.A., and Gliemann, J. (1992) J. Biol. Chem. 267, 14543-14546). Soluble recombinant urokinase receptor blocked the binding of pro-uPA to alpha 2MR/LRP but caused only a slight reduction in the affinity for binding of uPA.PAI-1. Moreover, pro-uPA bound to the urokinase receptor at the cell surface was not internalized and degraded unless activated to uPA and complexed with PAI-1. We conclude that pro-uPA is protected against degradation via alpha 2MR/LRP when bound to uPAR due to shielding of a binding contact in the A chain, whereas the affinity of uPAR-bound uPA.PAI-1 complex for binding to alpha 2MR/LRP remains sufficient to allow rapid internalization and degradation.

3T3 Cells↗
PubMed1994

Localization of vitronectin binding domain in plasminogen activator inhibitor-1.

Plasminogen activator inhibitor type 1 (PAI-1) is the rapid physiologic inhibitor of tissue-type plasminogen activator and urokinase-type plasminogen activator (uPA). In plasma and the extracellular matrix, PAI-1 is associated with the adhesive glycoprotein vitronectin. In order to characterize the PAI-1 structural domain responsible for binding to vitronectin, the segment of the PAI-1 cDNA encoding amino acids 13-147 (nucleotides 248-650) was randomly mutagenized and subcloned into a bacterial expression vector containing the mature PAI-1 coding sequence. Recombinant PAI-1 mutants were expressed in Escherichia coli and bacterial lysates assayed in duplicate for uPA inhibitory activity and vitronectin binding. Of 190 clones screened, six consistently demonstrated decreased vitronectin binding relative to uPA inhibitory activity. DNA sequence analysis of four of these clones identified 10 unique missense mutations, all located between base pairs 298 and 641, with each clone containing between one and four substitutions. Each substitution was expressed independently by site-directed mutagenesis and again analyzed for uPA inhibitory activity and vitronectin binding. Five point mutations that selectively disrupt vitronectin binding were identified. All 5 residues are located on the exterior of the PAI-1 structure. These findings appear to define a complex binding surface that bridges alpha-helices C and E to beta-strand 1A and includes amino acids 55, 109, 110, 116, and 123. These results suggest that vitronectin binding may stabilize the active conformation of PAI-1 by restricting the movement of beta-sheet A and thereby preventing insertion of the reactive center loop.

Amino Acid Sequence↗
PubMed1994

Engineering plasminogen activator inhibitor 1 mutants with increased functional stability.

Plasminogen activator inhibitor 1 (PAI-1), a member of the serine protease inhibitor (Serpin) superfamily, is the primary inhibitor of the plasminogen activators tPA and uPA. PAI-1 is produced in an active form but converts to an inactive or latent form with a half-life of approximately 1 h at pH 7.5, 37 degrees C. This study describes the construction, expression, and characterization of PAI-1 mutants with increased functional stability. Three mutations that disrupt an ion pair, present in latent PAI-1, between Arg-30 and Glu-350 (P4'), were introduced into recombinant PAI-1. All three mutant proteins maintained normal functional activity against both uPA and tPA. However, the half-life of each purified PAI-1 mutant was extended compared to the 1.1 h observed for wild-type PAI-1 (wtPAI-1) (1.2 h for Glu-350-->Arg, 2.0 h for Glu-350-->Pro, and 2.1 h for the Arg-30-->Glu mutation). An additional PAI-1 variant containing a second mutation designed to potentially reconstitute the ion pair (Arg-30-->Glu, Glu-350-->Arg) failed to restore the wild-type half-life. Circular dichroism spectra analysis indicated that the active and latent forms of wtPAI-1 and all four mutants contained similar secondary structural elements. Thermal stability determinations showed that latent wtPAI-1 was much more structurally stable than the active conformation. However, the latent form for all four mutants was significantly less stable than the corresponding wtPAI-1 conformer. This is the first report of PAI-1 mutants which have been specifically engineered to produce enhanced functional stability.

Base Sequence↗
PubMed1994

The urokinase receptor is expressed in invasive breast cancer but not in normal breast tissue.

We have studied expression of the urokinase receptor (u-PAR) in paraffin-embedded breast tissues at various stages of malignant progression. Forty-nine of 59 invasive cancers studied showed varying degrees of reactivity with our polyclonal antibody. The staining pattern was variable from case to case, although strong surface staining of tumor-associated macrophages was evident in most of these sections. In several cases, blood vessels in selected tumor areas were stained, as confirmed by treatment of adjacent sections with an anti-factor VIII antibody. These could represent regions of recent angiogenesis. Staining of tumor cells was observed in 21 of 59 cases and was extensive in 5 cases but confined to a small percentage of cells in the remaining 16 samples. In contrast with the cancer sections, all normal breast tissue (12 cases) was negative, as well as all fibroadenomas (4 cases), papillomas (5 cases), and hyperplasia with atypia (2 cases) studied. Seven carcinomas in situ examined were also negative for u-PAR, with the exception of few macrophages in two cases, suggesting that u-PAR expression may be associated with invasive tumor. The presence of u-PAR in human breast cancer and its absence from nonmalignant breast tissue supports the idea that plasminogen activation plays an important role in the process of cancer invasion. Expression of u-PAR on macrophages, endothelial cells, and cancer cells suggests the existence of complex paracrine interactions between tumor cells and stroma.

Base Sequence↗
PubMed1994

Immunologic detection of the cellular receptor for urokinase plasminogen activator.

The cellular receptor for urokinase plasminogen activator (uPA-R) is a monomeric phosphatidylinositol-linked glycoprotein (gp40-65) that may contribute to the invasive capacity of tumor and inflammatory cells by focusing the activity of urokinase (uPA) in converting plasminogen to plasmin, a serine protease capable of degrading extracellular matrix proteins. The further characterization of uPA-R has been facilitated by our recent development of a monoclonal antibody, anti-Mo3f, specific for uPA-R. This mAb bound to uPA-R expressed by phorbol myristate acetate-stimulated U-937 cells and by NIH-3T3 cells permanently transfected with uPA-R cDNA. In competitive binding assays, anti-Mo3f inhibited the binding of fluorescein-conjugated uPA ligand to uPA-R expressed by U-937 cells and uPA-R transfectants; conversely, preexposure of cells to saturating quantities of exogenous uPA partially blocked the subsequent binding of anti-Mo3f mAb to uPA-R. Anti-Mo3f mAb was employed as the capture reagent in an ELISA for the quantitation of soluble forms of uPA-R (derived from U-937 cells and recombinant uPA-R) which had a sensitivity of approximately 4-12 ng/ml. Anti-Mo3f mAb was also applied as a serologic probe for the detection of uPA-R expressed by human tumor tissues. By immunoperoxidase staining, anti-Mo3f demonstrated positive tumor cell staining in 4 of 16 breast and 7 of 31 prostate carcinomas in formalin-fixed, paraffin-embedded specimens. These data indicate that the anti-Mo3f mAb detects an epitope proximate to or within the ligand binding domain (domain 1) of uPA-R and may be useful as a tool for the serologic detection of uPA-R in soluble form or associated with human tumors.

3T3 Cells↗
PubMed1994

Immunomodulation by metals.

A symposium entitled Immunomodulation by Metals was held at the 32nd Annual Meeting of the Society of Toxicology (SOT) in New Orleans, Louisiana. The symposium was co-sponsored by the Immunotoxicology and Metals Specialty Sections of SOT and was designed to describe the types of adverse immunological reactions which occur in response to environmental and/or occupational exposure to metals. Epidemiological evidence and underlying mechanisms responsible for the observed alterations were also discussed. The following is a summary of each of the individual presentations.

Animals↗
PubMed1994

In vitro effects of hydroquinone, benzoquinone, and doxorubicin on mouse and human bone marrow cells at physiological oxygen partial pressure.

A comparative study was undertaken in order to assess the hematotoxic effects of hydroquinone (HQ), 1,4-benzoquinone (BQ), and doxorubicin (DX) on mouse and human bone marrow (BM) cells. Initial experiments indicated that the inhibitory effects of near-ambient pO2 and HQ on granulocyte/macrophage colony-stimulating factor (GM-CSF)-induced colony formation were additive. Thus, subsequent experiments were done under conditions of continuous toxicant exposure in complete medium at physiological temperature and O2 partial pressure. Viability was measured 24 hr after exposure, and at the concentrations tested, HQ was less cytotoxic than BQ. DX did not exhibit significant cytotoxicity at the concentrations used. Both HQ and BQ were slightly more cytotoxic to mouse BM cells than to human BM cells. Dose-response analyses of HQ, BQ, or DX inhibition of GM-CSF-induced proliferative and colony-forming responses indicated that murine GM progenitors were significantly less sensitive to HQ than to the majority of myeloid BM cells that proliferated in response to GM-CSF. This preferential resistance of GM progenitors to HQ was not observed when human BM cells were used. HQ was somewhat more inhibitory to human than to mouse GM-CSF responses. Inhibition of GM-CSF-induced responses by BQ correlated closely with cytotoxicity, and DX was 1000-fold more inhibitory to GM-CSF-induced proliferative and colony-forming responses than either HQ or BQ. Again, DX appeared to be slightly more inhibitory to human BM cells than to mouse BM cells. Purified human hematopoietic progenitor cells (HPCs) were also used in the dose-response analyses of HQ, BQ, or DX inhibition of GM-CSF-induced proliferative and colony-forming responses. Inhibition of GM-CSF-induced HPC responses by HQ, BQ, and DX was very similar to that obtained when BM mononuclear cells were used, suggesting that the human HPC is a target for the direct effects of HQ, BQ, and DX.

Adolescent↗
PubMed1994

Transforming growth factor-beta 1 enhances serum-induced dephosphorylation of the P53 protein in cell lines growth-inhibited by this factor.

A 24 hr TGF-beta 1 treatment (4 ng/ml) of SV40-transformed WI38 embryonic fibroblasts (VA13 cells) causes a moderate but reproducible inhibition of their serum-stimulated growth. By immunoprecipitation with the PAb122 antibody, we show that serum stimulation of previously serum-deprived cells causes a dephosphorylation of the wild type P53 protein, which is accentuated by the TGF-beta 1 treatment. The TGF-beta 1-enhanced dephosphorylation effect is also observed in two other cell lines growth-inhibited by TGF-beta 1, but which do not contain Large T (mink lung CCL64 and human KHOS cells). On the contrary, TGF-beta 1 treatment of the untransformed WI38 fibroblasts stimulates their growth, without affecting the phosphorylation of P53. Such treatment did not affect the expression of the corresponding mRNA nor the level of synthesis of the protein. The results suggest that the P53 protein could be a downstream target of TGF-beta 1 action on those cells growth-inhibited by the factor.

Animals↗
PubMed1993

Kinetics of inactivation of alpha-thrombin by plasminogen activator inhibitor-1. Comparison of the effects of native and urea-treated forms of vitronectin.

Kinetic studies are presented which show that native human vitronectin, but not urea-treated vitronectin, accelerates the inactivation of human alpha-thrombin by human plasminogen activator inhibitor-1 (PAI-1). We demonstrate that although urea-treated vitronectin binds PAI-1 with an affinity greater than that of native vitronectin, it does not accelerate the rate of inactivation of alpha-thrombin by PAI-1. We present evidence to suggest that the inability of urea-treated vitronectin to accelerate the reaction between alpha-thrombin and PAI-1 results at least in part from the inability of urea-treated vitronectin to bind to alpha-thrombin. The accelerated reaction between PAI-1 and alpha-thrombin can be accounted for by the formation of a tight complex between native vitronectin and PAI-1 that reacts in a saturable manner (Kd = 75 nM) with alpha-thrombin. The second-order rate constant (kI/Kd) for the reaction of the native vitronectin-PAI-1 complex with alpha-thrombin (1.64 x 10(5) M(-)-1 s-1) is 270-fold greater than the second-order rate constant for the reaction in the absence of vitronectin (610 m-1 s-1). The increase in the second-order rate constant is largely due to an increase in the affinity of alpha-thrombin for the native vitronectin-PAI-1 complex, as reflected by a greater than 25-fold reduction in the dissociation constant (Kd) observed for the vitronectin-PAI-1 complex relative to that of uncomplexed PAI-1.

Blood Proteins↗
PubMed1992

Strand-selective repair of DNA damage in the yeast GAL7 gene requires RNA polymerase II.

We examined the role of transcription in directing repair of DNA damage in active genes by comparing the repair of thymine glycols produced by H2O2 and of UV-induced pyrimidine dimers on each strand of the GAL7 gene of Saccharomyces cerevisiae. Repair of both thymine glycols and pyrimidine dimers on the transcribed strand of the gene occurs two to three times faster than on its nontranscribed strand or in the genome overall. When the gene is inactive, no preferential or strand-selective repair is observed. Using a yeast strain containing a temperature-sensitive mutation in one of the subunits of RNA polymerase II, we find that inactivating RNA polymerase II by shifting the cells to the nonpermissive temperature during repair eliminates the strand selectivity of repair under conditions where repair on the nontranscribed strand of the gene and in the genome overall are only slightly affected. Our observation of strand-selective repair of thymine glycols in the GAL7 gene is the first evidence that this repair process occurs for a nonbulky lesion. In addition, we demonstrate that the transcriptional complex plays a critical role in directing repair to the transcribed strand of active genes.

Blotting, Southern↗
PubMed1992

Saturation mutagenesis of the plasminogen activator inhibitor-1 reactive center.

Plasminogen activator inhibitor-1 (PAI-1) is a specific inhibitor of the serine proteases tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA). To systematically investigate the roles of the reactive center P1 and P1' residues in PAI-1 function, saturation mutagenesis was utilized to construct a library of PAI-1 variants. Examination of 177 unique recombinant proteins indicated that a basic residue was required at P1 for significant inhibitory activity toward uPA, whereas all substitutions except proline were tolerated at P1'. P1Lys variants exhibited lower inhibition rate constants and greater sensitivity to P1' substitutions than P1Arg variants. Alterations at either P1 or P1' generally had a larger effect on the inhibition of tPA. A number of variants that were relatively specific for either uPA or tPA were identified. P1Lys-P1'Ala reacted 40-fold more rapidly with uPA than tPA, whereas P1Lys-P1'Trp showed a 6.5-fold preference for tPA. P1-P1' variants containing additional mutations near the reactive center demonstrated only minor changes in activity, suggesting that specific amino acids in this region do not contribute significantly to PAI-1 function. These findings have important implications for the role of reactive center residues in determining serine protease inhibitor (serpin) function and target specificity.

Amino Acid Sequence↗
PubMed1992

Effects of 2-mercaptoethanol and buthionine sulfoximine on cystine metabolism by and proliferation of mitogen-stimulated human and mouse lymphocytes.

Cysteine is an essential amino acid for lymphocytes and its anabolic products are intimately involved in lymphocyte activation. The purpose of this study was to assess the uptake and subsequent utilization of cyst(e)ine by mitogen-stimulated human peripheral blood mononuclear cells (PBMC), to evaluate the effect of an exogenous thiol, 2-mercaptoethanol (2ME), on these processes, and to compare human and mouse lymphocyte reactivities. Unlike mouse lymphocytes, the proliferation of human T-cells was inhibited by addition of 2ME although 2ME enhanced cystine uptake. Optimal responses to T-cell mitogens (Con A and PHA) were obtained with a cystine concentration of greater than or equal to 25 and 200 microM for human and mouse cells, respectively, and 2ME enhanced DNA synthesis of Con A-stimulated mouse cells regardless of the cystine dose; however, 2ME enhanced the response of human cells only in the presence of suboptimal doses of cystine. To assess whether 2ME's inability to enhance human PBMC responses was related to their glutathione (GSH) content, the human PBMC were pretreated with buthionine sulfoximine (BSO, an inhibitor of GSH synthesis). Even when the initial intracellular GSH concentration was lowered to below that of mouse lymphocytes, 2ME still inhibited proliferation. In contrast, addition of 2ME to human PBMC maintained in the presence of BSO enhanced the proliferative response suggesting that a critical level of thiols is needed for proliferation. The ability of 2ME to enhance proliferative responses in cystine deficient medium supports this contention. Consistent with thiol involvement in activation, Con A increased [35S]cystine uptake 2-fold within 4 h of incubation and enhanced subsequent conversion of cystine into cysteine and GSH. Interestingly, BSO treatment only slightly inhibited Con A-induced protein synthesis (5%), but it significantly suppressed conversion of cystine into cysteine or GSH (80-95%) and blocked DNA synthesis (90%). Overall, the results indicate that various differential thiol characteristics must exist between human and mouse lymphocytes and that a reducing equivalent is necessary for DNA synthesis but not lymphocyte activation.

Adolescent↗
PubMed1991

The oxidative inactivation of plasminogen activator inhibitor type 1 results from a conformational change in the molecule and does not require the involvement of the P1' methionine.

Plasminogen activator inhibitor 1 (PAI-1) is sensitive to oxidative inactivation, and it has been suggested that specific oxidation of a methionine residue, Met347, situated in the P1' position of the reactive center may be the cause of the inactivation. To test this hypothesis we have purified and biochemically characterized mutant proteins of PAI-1 in which Met347 and either of two other methionines, Met266 or Met354, has been replaced with oxidation-resistant valine residues. The mutant proteins were found to be equally sensitive to oxidation as wild-type PAI-1, suggesting that a specific oxidation of the P1' Met347 is not responsible for the inactivation. When PAI-1 was oxidized, circular dichroism analysis revealed a rapid conformational change that correlated to the loss of inhibitory activity. The oxidation sensitivity of PAI-1 was enhanced dramatically in the presence of 0.001% sodium dodecyl sulfate, and the circular dichroism spectrum was significantly different from that of untreated PAI-1, suggesting that the increased sensitivity to oxidation may be caused by a conformational change in the inhibitor molecule. Taken together, our data suggest that the oxidative inactivation of PAI-1 is not caused by the specific oxidation of the P1' methionine but results from a conformational change in the protein structure.

Base Sequence↗