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Biomedical subjects

D L Emerson

Publications and source records attributed to D L Emerson.

At least 19 recordsLinked to original sources
PubMed1995

Synthesis, topoisomerase I inhibitory activity, and in vivo evaluation of 11-azacamptothecin analogs.

A series of analogs based on a novel template, 11-aza-(20S)-camptothecin, were obtained from total synthesis and tested as potential anticancer drugs in the topoisomerase I enzyme cleavable complex assay. The parent compound 11-aza-(20S)-camptothecin (8) was derived from a Friedlander condensation between the known aminopyridine derivative 3-(3-amino-4-picolylidene)-p-toluidine and optically active tricyclic ketone 7. Compound 8 had activity approximately twice that of (20S)-camptothecin in the calf thymus topoisomerase I cleavable complex assay. Compounds were prepared wherein the 11-aza nitrogen atom was quaternized as either the corresponding N-oxide or methyl iodide. Compounds with quaternized N-11 showed improved water solubility and were equipotent to the clinically investigated camptothecin analog topotecan in the cleavable complex assay. These compounds were evaluated in vivo in nude mice bearing HT-29 human colon carcinoma xenografts. The analog 11-aza-(20S)-camptothecin 11-N-oxide was found to significantly retard tumor growth when compared to untreated controls. Finally, 7,10-disubstituted 11-azacamptothecin analogs were synthesized using Pd(0) coupling reactions of 10-bromo-7-alkyl-11-aza-(20S)-camptothecins 19 and 20, which in turn were available from a Friedlander condensation of the novel bromopyridine derivatives 17a and 17b with 7. Among the 10-substituted series, a number of analogs displayed extremely high in vitro potency against topoisomerase I and improved aqueous solubility. A significant number of the compounds were found to be active in whole cell cytotoxicity assays and several were evaluated in nude mice bearing the HT-29 tumor xenografts. The most effective of these proved to be (S)-11-aza-7-ethyl-10-(aminohydroximinomethyl)camptothecin trifluoracetic acid salt (27), a potent topoisomerase I inhibitor which demonstrated excellent efficacy in both short term and in extended in vivo assays. A comparison between in vitro enzyme data and in vivo data from nude mouse studies in other compounds in this series revealed a poor overall correlation between topoisomerase inhibition in vitro and antitumor efficacy in vivo.

Animals↗
PubMed1995

Synthesis and antitumor activity of novel water soluble derivatives of camptothecin as specific inhibitors of topoisomerase I.

The synthesis and antitumor activities of the novel water soluble camptothecin derivatives 7-[(4-methylpiperazino)methyl]-10,11-(methylenedioxy)-(20S)-campto thecin trifluoroacetate (6) and 7-[(4-methylpiperazino)methyl]-10,11-(ethylenedioxy)-(20S)-camptot hecin trifluoroacetate (7) are described. The solubilities of compounds 6 and 7 were measured to be 4.5 and 5.8 mg/mL, respectively, in pH 5 acetate buffer in contrast to < 0.003 mg/mL for camptothecin in the same buffer. In the purified topoisomerase I cleavable complex enzyme assay, compounds 6 and 7 demonstrated potent inhibition of topoisomerase I with IC50's of 300 and 416 nM, respectively, in comparison to 679 nM for camptothecin and 1028 nM for topotecan. In human tumor cell cytotoxicity assays, compounds 6 and 7 demonstrated potent antitumor activity against ovarian (SKOV3), ovarian with upregulated MDRp-glycoprotein (SKVLB), melanoma (LOX), breast (T47D), and colon (HT29) with IC50's ranging from 0.5 to 102 nM. Compounds 6 and 7 induced tumor regressions in the HT29 human colon tumor xenograft model and demonstrated similar rank order of potency compared to in vitro assay results.

Animals↗
PubMed1995

In vivo antitumor activity of two new seven-substituted water-soluble camptothecin analogues.

The development of camptothecin-like compounds as inhibitors of topoisomerase I for the treatment of resistant tumors has generated clinical excitement in this new class of drugs. We have developed two novel water-soluble camptothecin analogues which are specific inhibitors of topoisomerase I and are potent cytotoxins with significant antitumor activity. We added water-solubilizing groups off position 7 in the B ring of either 10,11-ethylenedioxy- or 10,11-methylenedioxy-20(S)-camptothecin. These water-soluble camptothecin analogues were demonstrated to be nanamolar inhibitors of the topoisomerase I enzyme in the cleavable complex assay. The compounds, GI147211 [7-(4-methylpiperazinomethylene)-10,11-ethylenedioxy-20(S)-camp tot hecin], and GI149893 [7-(4-methylpiperazinomethylene)-10,11-methylenedioxy-20(S)-cam pto thecin], were compared to topotecan, a known water-soluble inhibitor of topoisomerase I. Both GI compounds were found to be slightly more potent than topotecan as inhibitors of topoisomerase I in the cleavable complex assay and were 1.5-2 times more soluble. Tumor cell cytotoxicity assays using 5 separate cell lines demonstrated that both GI compounds were 5-10 times more potent than topotecan, although by comparison all three topoisomerase I inhibitors were unaffected by the multidrug resistance P-glycoprotein. The antitumor activity of all three topoisomerase I inhibitors was compared concomitantly in two human colon xenograft models. In both models, GI147211 and GI149893 were able to induce regression of established HT-29 and SW-48 colon tumors by as much as 60%. The antitumor activity of both compounds were also demonstrated in the MX-1 and PC-3 xenografts. Microscopic examination of selected tissues indicated that drug-induced toxicity was primarily limited to the gastrointestinal tract and was comparable among the three compounds. Further clinical development of this class of compounds is ongoing.

Animals↗
PubMed1990

Reconstituted basement membrane enhances neurite outgrowth in PC12 cells induced by nerve growth factor.

Rat pheochromocytoma cells (PC12) respond to nerve growth factor (NGF) by elaborating neurites. We have studied the effect of extracellular matrix proteins on responsiveness of PC12 cells to NGF. NGF elicited neurite outgrowth in cells plated on collagen, fibronectin, or laminin within 48-72 h. In contrast, cells entrapped in reconstituted basement membrane prepared from the Engelbreth-Holm-Swarm tumor (EHS-BM) responded within 24 h. Cells plated on collagen or fibronectin required a minimal dose of 50 ng/ml NGF to achieve the same effect as cells entrapped in EHS-BM at 1 ng/ml NGF. Neurites displayed by cells plated in EHS-BM were more extensively branched and remained intact up to 21 days following a single administration of NGF.

Adrenal Gland Neoplasms↗
PubMed1988

Western blot analysis of serum antibody reactivity with human melanoma cell antigens in alopecia areata and vitiligo.

Antibody reactivity to melanocyte-derived cells was investigated in patients with alopecia areata or totalis by use of Western blot analysis of detergent-solubilized membrane antigens of a human melanoma cell line, M14. Reactivity was detected in the sera of 9 of 27 alopecia areata or totalis patients, 8 of 13 vitiligo patients, and 6 of 24 normal control subjects. Significant differences between patient and control sera were found in the number and distribution of antibody specificities detected. In vitiligo sera, there was an increased prevalence of reactivity to a melanoma antigen of 52,000 mol wt. In contrast, the predominant specificities in alopecia areata sera were for antigens of 74,500 and 70,800 mol wt, and the majority of positive sera were from patients with total hair loss. These findings suggest that autoreactivity to pigmented cells occurs in certain patients with alopecia areata or totalis.

Alopecia↗
PubMed1987

High levels of group-specific component (vitamin-D-binding protein) in the cerebrospinal fluid of infants aged less than 2 months.

Because of the possible involvement of group-specific component (Gc) or vitamin-D-binding protein in the immunological functions of mononuclear cells and the increased risk of central nervous system infections in early infancy, we studied Gc levels in the cerebrospinal fluid (CSF) of children. CSFs were examined for the Gc concentration using ELISA and rocket immunoelectrophoresis, with purified Gc as standard. The results showed a significant inverse correlation (p less than 0.05) between the age of the patients and CSF Gc levels. Gc levels in the CSF were significantly increased in infants less than 2 months of age (12.5 micrograms/ml), as compared to infants greater than 2 months (1.7 micrograms/ml, p less than 0.0028). In children greater than 2 months of age a significant correlation was found between Gc levels and those of other CSF proteins (albumin, IgG and total protein, p less than 0.002). However, no significant correlation between Gc levels and those of other CSF proteins was apparent in infants less than 2 months of age, indicating the possibility that the concentration of Gc in the CSF may be selectively increased in this age group.

Aging↗
PubMed1987

Correlation between extent of liver damage in fulminant hepatic necrosis and complexing of circulating group-specific component (vitamin D-binding protein).

Increased complexing of circulating group-specific component (Gc, vitamin D-binding protein) has been found in humans under conditions of presumed increased actin release, and particularly fulminant hepatic necrosis (FHN). We therefore used a hamster model of acetaminophen-induced FHN to further investigate this phenomenon. Liver damage was monitored by aspartate aminotransferase (AST) activity and by histologic examination, and serum Gc was analyzed by polyacrylamide gel electrophoresis (PAGE) and transblotting with anti-Gc. In controls and treated animals displaying slight liver damage, greater than 90% of Gc was of mobility corresponding to native purified hamster Gc, whereas with more severe liver damage up to 100% of Gc was found in one of two cathodal configurations that appear to correspond to complexes with actin. Densitometric quantitation of complexed Gc demonstrated a strong correlation with the severity of liver damage. These results show PAGE to be a useful method of estimating the percentage of Gc complexed in serum, and suggest that cell damage may be followed by the appearance in the circulation of cellular actin that complexes with Gc.

Actins↗
PubMed1986

Group-specific component (vitamin D binding protein) prevents the interaction between G-actin and profilin.

Profilin purified from human platelets formed a 1:1 molar ratio complex with rabbit skeletal muscle G-actin but was displaced by purified serum Gc (vitamin D binding protein) in a dose-dependent fashion as assessed by chromatography and ultrafiltration. This suggested that Gc and profilin competed for the same binding area on G-actin, with Gc-G-actin complexes being more stable than profilin-G-actin complexes in vitro. The binding domain for Gc on G-actin was localized to a 16,000-Da C-terminal fragment of G-actin generated by Staphylococcus aureus V8 protease, as judged by comigration on two-dimensional electrophoresis and also by overlaying electrophoresis gels with 125I-Gc. Previous studies have reported that residues 374 and 375 of G-actin are essential for binding of profilin. In this study, experiments involving tryptic removal of Cys-374 labeled with the fluorescent probe N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)-ethylenediamine showed that these C-terminal amino acids were not necessary for interaction with Gc.

Actins↗
PubMed1986

Gc (vitamin D-binding protein) binds the 33.5 K tryptic fragment of actin.

Limited proteolysis of G-actin was performed with trypsin and chymotrypsin to compare the binding sites for Gc and DNase. DNase I bound to the N-terminal area corresponding to the major cleavage site on G-actin (residues 62-68) and inhibited proteolysis, but did not bind the 33.5K C-terminal fragment (G-actin33.5) generated. In contrast, Gc did not exert any inhibitory effect upon proteolysis of the intact native G-actin42.0 molecule, although its presence protected G-actin33.5 from further proteolysis. This was shown by gel filtration to be due to the formation of complexes between Gc and G-actin33.5.

Actins↗
PubMed1985

Accurate quantitation of native Gc in serum and estimation of endogenous Gc: G-actin complexes by rocket immunoelectrophoresis.

Complex formation between purified Gc and G-actin caused increased rocket height on immunoelectrophoresis with monospecific Gc antiserum, and artifactually high calculated Gc levels. The increase in rocket height varied in log: linear fashion with the amount of G-actin present, up to a plateau attained at equimolarity. The raw Gc values could therefore be corrected to within +/- 10% of known levels by addition of excess G-actin and use of standard plots obtained with Gc after saturation with G-actin. This also allowed quantitation of the percentage of Gc complexed with G-actin. In subsequent studies of whole human sera, comparison of normal controls with pregnant subjects and patients with liver disease showed evidence of differences both in absolute quantities of Gc and the relative proportion circulating as complex with G-actin. This appeared to be due to increased release of cellular actin into the extracellular space. These results show that rocket immunoelectrophoresis can be modified to provide accurate Gc levels, and also information concerning different molecular forms of this protein.

Actins↗
PubMed1985

Distinct sites on the G-actin molecule bind group-specific component and deoxyribonuclease I.

Addition of group-specific component (Gc) to G-actin with or without deoxyribonuclease I (DNAase) led to formation of binary complexes (Gc-G-actin) and ternary complexes (Gc-G-actin-DNAase) respectively. The electrophoretic mobility of ternary complexes, as shown by crossed and rocket immunoelectrophoresis, was slower than that of binary complexes, although both were faster than native Gc. In gradient polyacrylamide-gel electrophoresis, such complexes could again be resolved, apparently on the basis of relative molecular size: Gc-G-actin-DNAase (Mr approx. 131000), Gc-G-actin (Mr approx. 98000) and Gc (Mr approx. 56000). In contrast, the pI of ternary complex was indistinguishable by isoelectric focusing from that of binary complex, even though both were clearly more acidic than native Gc. The affinity of Gc for G-actin (affinity constant, Ka, 1.9 X 10(8) M-1) was not significantly altered by additional interaction with DNAase (Ka, 1.5 X 10(8)M-1), and both binary and ternary complexes still bound 25-hydroxycholecalciferol. In addition, the inhibitory effect of G-actin on DNAase activity was not discernibly affected by interaction with Gc. These results demonstrate that the various molecular forms of Gc can be distinguished by physicochemical parameters, and that Gc and DNAase bind to distinct sites on G-actin and can interact both independently and contemporaneously with this molecule.

Actins↗
PubMed1985

Structural studies of T lymphocyte Fc receptors. Association of Gc protein with IgG binding to Fc gamma.

To obtain further information concerning the structure of Fc IgG-binding sites on human peripheral blood lymphocytes, enriched T-cells were surface-radioiodinated and treated with nonionic detergent, and the soluble supernatant was submitted to affinity chromatography selecting for components binding complexed IgG. Analysis of eluted material by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, isoelectric focusing, and two-dimensional electrophoresis demonstrated the major proteins to be of Mr 56,000, pI 4.8-5.1 and Mr 60,000, pI 5.0-5.6, and these were radiolabeled, indicating an origin in part from the T-cell membrane. While the Mr 56,000 band gave positive reactions upon transblotting with antisera to Gc protein, the identity of the Mr 60,000 protein remains unknown. Three other components were detected, although with less consistency; Mr 78,000, 42,000, and 20,000-25,000, respectively. Immunocytochemical experiments showed that less than 5% freshly isolated native T-cells were positive with antiserum to human Gc, but, after IgG antibody-coated erythrocyte rosetting, the number of positive cells increased to 15-25%, in close agreement with the percentage of IgG antibody-coated erythrocyte rosette-positive T-cells. These findings therefore indicate that, in addition to interaction with components of Mr 60,000, 42,000, and 20,000-25,000, complexed IgG binding to Fc gamma of human peripheral blood T-cells also becomes spatially associated with Gc protein.

Chromatography, Affinity↗
PubMed1985

Altered configuration of Gc on the plasma membrane of transformed and malignant human B lymphocytes.

Normal human peripheral blood B cells exhibit strong membrane fluorescence for Gc (vitamin D-binding protein), and this protein can form a close spatial relationship with integral membrane immunoglobulin (mIg) with evidence of codistribution in the lipid bilayer. In contrast, fluorescence for both Gc and mIg has been found in this study to be weak or absent in several B lymphoblastoid cell lines and in chronic lymphocytic leukemia B cells. Moreover, the comobility of these components, where detectable, was also impaired. In abnormal B cells, the intensity of membrane fluorescence for Gc was substantially increased after crosslinking of mIg with antibody, and the latter was also associated with increased specific radioiodination of Gc by lactoperioxidase. These results indicate that Gc can apparently become displaced under certain circumstances within or through the lipid bilayer. The altered content or membrane topography of Gc in such abnormal B cells might be associated with impaired expression and mobility of mIg.

B-Lymphocytes↗
PubMed1985

Presence of Gc (vitamin D-binding protein) and interactions with actin in human placental tissue.

The distribution of Gc protein and actin and their interactions were studied in normal full-term human placentae. Both Gc and actin were detected by physicochemical analysis of isolated trophoblast membranes. Immunofluorescence of native placental sections showed fluorescence for both proteins on smooth muscle cells lining the fetal stem vessels, intervillous fibrin, villous fibrinoid, trophoblast membrane, and cytoplasm of villous stromal cells. Binding of Gc was demonstrated by prior incubation of sections with purified Gc which led to a striking increase in intensity of Gc fluorescence, but actin fluorescence was unaffected by this procedure and by preincubation with actin. Endogenous Gc and actin could also be removed by washing of tissue sections with chaotrope--3 M KCl or 3 M NH4SCN, denaturant--6 M urea, or glycine-HCl pH 3.8, as judged by fluorescence and SDS-PAGE of the wash supernatant. Phenotypic analysis of Gc eluted from trophoblast membranes and of corresponding matched maternal and fetal cord sera by isoelectric focusing indicated that trophoblast Gc was of predominantly maternal origin. Although the roles of Gc and actin in the placenta are unknown, these results indicate that Gc may be another maternal protein for which specific binding sites are expressed on the membrane of placental trophoblast.

Actins↗
PubMed1984

The genetic variants of group-specific component (vitamin D-binding protein) possess different binding characteristics for immobilized Cibacron Blue 3-GA.

The elution profiles of several variants of the Gc protein have been studied after chromatography on immobilized Cibacron Blue 3-GA. The allele products belonging to the Gcl type were retarded and eluted with a Ve/Vo at 1.5, as previously reported for the Gcl-1 phenotype [Chapuis-Cellier, Gianazza & Arnaud (1982) Biochim. Biophys. Acta 709, 353-357]. The allele products belonging to the Gc2 type were further retarded (Ve/Vo at 2.6), and both Gcl and Gc2 allele products were clearly separated in heterozygous individuals. This observation allows the isolation and purification of Gc variants in heterozygous individuals which carry the combination Gcl variant-Gc2, Gcl-Gc2 variant, or Gcl variant-Gc2 variant. In contrast, the corresponding holoproteins did not bind to the gel and were eluted in the void volume. This suggests that the interaction of Gc with immobilized Cibacron Blue 3-GA involves the binding site of the protein for 25-hydroxycholecalciferol and that the dye behaves as a 'pseudoligand' for the protein. In addition, our data suggest that the different elution profiles of the variants could reflect a different affinity of the gene products for the dye.

Alleles↗
PubMed1984

Gc (vitamin D binding protein) binds to cytoplasm of all human lymphocytes and is expressed on B-cell membranes.

Peripheral blood lymphocytes were examined immunohistologically for evidence of interactions with Gc protein, a major vitamin D binding protein in serum. In the cytoplasm, binding sites for purified Gc were readily detectable in all cells, and these sites were only partially occupied by Gc. In contrast, on the membrane of viable cells, there was negligible evidence of binding of either the apo- or holoform of Gc protein, but substantial quantities of firmly bound immunoreactive endogenous Gc were detected. Separation experiments and double-label fluorescence with antisera recognizing defined phenotypic markers showed immunoreactive membrane Gc on 30-40% of unfractionated mononuclear cells and greater than 95% of monocytes or B cells. Only 5-8% of T cells were similarly reactive; these were not apparently confined to any given subset. Extraction of unfractionated cells with 6 M urea or solubilization in Nonidet P-40 released immunoreactive Gc protein, with physicochemical properties indistinguishable from those of Gc purified from serum (apparent MW 56K; pI 4.8-5.1). These findings indicate that membrane Gc may represent another surface immunofluorescence marker for B cells, and may play a role in immunocyte function.

B-Lymphocytes↗