Biomedical subjects
Publications and source records attributed to B E Miller.
Descriptions of derivatization reactions applied to high-performance liquid chromatography for pharmaceuticals classified as alkaloids, amines, antibiotics, barbiturates, carbonyl/carboxylic compounds, catecholamines, hydroxy compounds, steroids, sulfur compounds, and miscellaneous are summarized.
Type II cells isolated from the lungs of rats exposed to silica can be separated into two populations by using centrifugal elutriation. One population, designated type IIA, appears similar to type II cells isolated from control lungs. The second population, designated type IIB, consists of type II cells that are larger than type IIA cells or control type II cells. In addition, the type IIB (or hypertrophic) cells contain lamellar bodies which are larger and more numerous than lamellar bodies in type IIA or control type II cells. After centrifugal elutriation, the type II cell populations were purified by differential adherence on rat IgG-coated culture dishes for biochemical and metabolic studies. Type IIB cells contained elevated amounts of protein and total RNA in comparison to type IIA and control type II cells. Type IIB cells contained approximately 2.5-fold more phospholipid than control type II cells (51.5 +/- 3.0 micrograms/10(6) cells versus 20.5 +/- 4.1 micrograms/10(6) cells). Incorporation of the phospholipid precursors [14C]choline and [3H]palmitate into cellular phosphatidylcholines was also increased in type IIB cells. After 120 minutes of incubation, incorporation of [14C]choline into total phosphatidylcholine by type IIB cells was 250% greater than in controls; at this same time point, incorporation of [3H]palmitate was approximately 150% greater than in controls. Incorporation of [14C]choline into disaturated phosphatidylcholine by type IIB cells was 200% greater than in controls. However, no increased incorporation of [3H]-palmitate into disaturated phosphatidylcholine by type IIB cells was seen. These results suggest that the hypertrophic type II cell is responsible for the increases in surfactant-associated phospholipids in the lungs of rats exposed to silica.
Human cerebrospinal fluid (CSF) contains many uncharacterized endogenous opioids, in addition to the known enkephalins, endorphins, and dynorphins. These opioids may be separated by gel filtration chromatography and identified by radioreceptor assay for opioid activity. One region of the chromatographic elution profile, designated "Peak B" has previously been shown to be related to the pain status of chronic pain patients. We now report that human Peak B isolated from the CSF of pain-free elective surgery patients is present at a typical concentration equivalent in activity to 1.4 pmol of morphine sulfate per ml of CSF measured by radioreceptor assay. At a dose of 0.06 and 0.12 pmol morphine sulfate equivalents of CSF (MSE), injected into the cerebroventricular system of the mouse, Peak B produced an antinociceptive effect, the intensity and duration of which was dose-dependent and which was antagonized by naloxone. The mouse vas deferens (MVD) preparation was inhibited by Peak B in a manner that was sensitive to antagonism by naloxone only at low (less than 1.0 microM) but not at higher (greater than 6.0 microM) concentrations of the antagonist. Peak B activity in the MVD assay was unaffected by treatment with trypsin or alpha-chymotrypsin.
Three nested models describing the growth of individual subpopulations in a heterogeneous environment are described. The models represent the dynamics of two populations which compete, to varying degrees, for common resources. The first model describes growth in a totally non-competitive micro-environment, the second model describes an ecology in which competition is proportional to competitor population size, and the third model ecology extends the model described by Jansson & Revesz (1974), which allows one population to emerge from the other. The critical points for each model are defined using the isoclines derived from the Ordinary Differential Equations (ODE's) describing competitive growth. The critical points for each model are characterized by the signs of the eigenvalues of the variational matrix at each point. The theoretical results of the analysis show that a competitive model ecology with Verhulstian logistics allows four critical points: the origin which is a repeller, two competitive exclusion points, and an equilibrium state (Waltman, 1983). The extended model ecology of Jansson & Revesz (1974), allows three critical points: the origin which is a repeller, competitive exclusion of the first population, and an equilibrium point. Data from a human adenocarcinoma of the colon and murine mammary tumors are used as qualitative measures of the dynamics of the three micro-ecologies. Issues such as stochastic extension to model small populations either for clonal extinction or heterogeneous emergence are discussed.
To better define the biologic function of the type II insulin-like growth factor (IGF) receptor, we raised a blocking antiserum in a rabbit by immunizing with highly purified rat type II IGF receptor. On immunoblots of crude type II receptor preparations, only bands corresponding to the type II IGF receptor were seen with IgG 3637, indicating that the antiserum was specific for the type II receptor. Competitive binding and chemical cross-linking experiments showed that IgG 3637 blocked binding of 125I-IGF-II to the rat type II IGF receptor, but did not block binding of 125I-IGF-I to the type I IGF receptor, nor did IgG 3637 block binding of 125I-insulin to the insulin receptor. In addition, IgG 3637 did not inhibit the binding of 125I-IGF-II to partially purified 150- and 40-kDa IGF carrier proteins from adult and fetal rat serum. L6 myoblasts have both type I and type II IGF receptors. IGF-I was more potent than IGF-II in stimulating N-methyl-alpha-[14C]aminoisobutyric acid uptake, 2-[3H]deoxyglucose uptake, and [3H]leucine incorporation into cellular proteins. IgG 3637 did not stimulate either 2-[3H]deoxyglucose uptake, N-methyl-alpha-[14C]aminoisobutyric acid uptake, or [3H]leucine incorporation into protein when tested alone. Furthermore, IgG 3637 at concentrations sufficient to block type II receptors under conditions of the uptake and incorporation experiments did not cause a shift to the right of the dose-response curve for stimulation of these biologic functions by IGF-II. We conclude that the type II IGF receptor does not mediate IGF stimulation of N-methyl-alpha-[14C]aminoisobutyric acid and 2-[3H]deoxyglucose uptake and protein synthesis in L6 myoblasts; presumably, the type I receptor mediates these biologic responses. The anti-type II receptor antibody inhibited IGF-II degradation in the media by greater than 90%, suggesting that the major degradative pathway for IGF-II in L6 myoblasts utilizes the type II IGF receptor.
Using an assay based on polypeptide mobility in one- and two-dimensional polyacrylamide electropherograms, we purified the Salmonella typhimurium cysB protein from an Escherichia coli strain carrying plasmid pGBK14, in which the S. typhimurium cysB gene is under transcriptional control of the strong promoter PL from phage lambda. cysB protein constitutes approximately 4% of total soluble protein in such cells and was obtained with good yield at a purity of 85% after precipitation of nucleic acids with streptomycin sulfate, ammonium sulfate fractionation, and hydrophobic chromatography on methyl agarose. Material with 95% purity was obtained with poor yield by ion-exchange chromatography of native protein and with good yield by size exclusion chromatography of denatured protein in sodium dodecyl sulfate. Physicochemical studies of the purified cysB protein show that it is a tetramer of identical 36-kDa subunits with a pI, amino acid composition, amino-terminal sequence, and carboxyl-terminal amino acid content identical to those known from previous studies or deduced from the cysB DNA sequence. Although O-acetyl-L-serine is believed to bind to cysB protein to form a complex that stimulates transcription of genes of the cysteine regulon, we were unable to demonstrate such an interaction using methods that would have detected binding with a Kd of less than 0.1 mM.
Thrombin activation of platelets induces the release of a high molecular weight glycoprotein, thrombospondin. On treatment with factor XIII transglutaminase and [3H]putrescine, thrombospondin undergoes specific incorporation of this labeled amine, with 2-3 mol of putrescine being incorporated per mol of thrombospondin. Analysis of plasmin digests of [3H]putrescine-thrombospondin showed that the Mr 53,000-core peptide contains the glutamine site for amine incorporation. In the absence of amine substrate, thrombospondin was found to provide both donor (glutamine) and acceptor (lysine) sites for intermolecular cross-links by factors XIIIa, and high molecular weight protein complexes were formed. Homopolymers of thrombospondin were also observed by electron microscopy. Thrombin-cleaved thrombospondin has more cross-linking sites accessible for [3H]putrescine incorporation or for cross-linkage to itself than does the uncleaved native protein. Examination of thrombospondin cross-linkage in the presence of other protein substrates (fibronectin, collagen, fibrinogen, and von Willebrand factor) for factor XIIIa, resulted in reduced thrombospondin polymer formation. Electron microscopy and autoradiography of fibrin clots formed in the presence of 125I-thrombospondin showed an association of thrombospondin with fibrin fibrils. However, confirmation that this association involves covalent epsilon-(gamma-glutamyl)lysyl cross-links between thrombospondin and fibrin was not obtained.
The pharmacology of aminoglutethimide (AG) was studied in two subsequent trials without hydrocortisone supplementation. A total of 79 patients with metastatic breast cancer entered the study, and their plasma and urine samples were analyzed by high-performance liquid chromatography (HPLC). Thirty evaluable patients with a median age of 57 years (range, 37-79) were treated with the standard dose of 1000 mg/day, and 37 evaluable patients with a median age of 59 years (range, 35-79) received 500 mg/day. The median follow-up in the two groups was 5 months (range, 1-16) and 4 months (range, 1-21), respectively. After the first oral dose of 500 mg, peak plasma concentrations of AG were observed 1-4 h after administration in 15 patients. The elimination half-life was 10.1 +/- 1.7 h (mean +/- SD) after initial dosage; it decreased significantly to 6.9 +/- 1.2 h after 8 weeks of treatment. The area under the curve of AG concentrations was 92.5 +/- 14.2 micrograms/ml x h. The total clearance rate was 5.5 +/- 0.9 1/h and the volume of distribution was 80 +/- 111. About 23% of the drug was excreted unchanged in the urine. The major metabolite, N-acetyl-AG (AAG), had the same half-life as AG. A comparison on day 7 of treatment revealed that doses of 1000 and 500 mg yielded AG plasma concentrations of 9.0 +/- 1.2 and 4.5 +/- 0.5 micrograms/ml, respectively. After 1 month of treatment, however, AG plasma levels of 6-7 and 4-5 micrograms/ml were observed, respectively. A 50% reduction of dose, therefore, resulted in only 30% lower AG levels during continuous treatment. Apparently, the induction of metabolism is of greater importance in standard-dose than in lower dose treatment. The plasma concentrations of AG did not bear a relationship to the clinical response.
In order to quantitate the effects of tumour subpopulation interactions, we have devised a method to determine the subpopulation composition of tumours by using paired tumour cell lines able to grow in different selective media. Line 4T07 forms colonies in thioguanine but not in HAT and line 168 forms colonies in HAT but not in thioguanine. An independent technique of determining tumour cell content was used to validate this method: line 168 and 4T07 cells are distinguishable by flow cytometry after staining with propidium iodide for DNA content. Mixtures of cell suspensions prepared from each unmixed tumour, as well as from tumours arising from mixtures of these lines, were analysed by both the colony formation assay and by the DNA content assay. The colony formation assay yielded values in good agreement with the DNA content assay, but was considerably more sensitive in that it was able to quantitate minority subpopulations that constituted less than 10% of the tumour. Both methods revealed that in tumours arising from mixtures, the tumour cells were almost entirely line 4T07, even when the inoculum had contained a high proportion of 168 cells. Since line 168 cells are very tumorigenic per se, these results suggest that line 4T07 cells are capable of interfering with 168 proliferation in mixed tumours, either directly or through a host-mediated mechanism.
The interaction of human thrombospondin with mucopolysaccharides has been measured quantitatively. Binding of thrombospondin to heparin was examined utilizing an assay employing an 125I-labeled LMW heparin glycosaminoglycan (Mr = 8500). By this means, a class of binding sites was detected that bound approximately 2 moles of LMW heparin per mole of thrombospondin with a Kd of 2.4 nM. The binding stoichiometry of LMW heparin to thrombospondin was confirmed by fluorescence polarization experiments in which thrombospondin bound 3 moles of fluorescamine-labeled LMW-heparin mole protein. A variety of mucopolysaccharides were able to inhibit the interaction of thrombospondin with 125I-LMW heparin, the most effective being heparin sulfate and dermatan sulfate. However, PF4 was found to be an even more potent inhibitor, approximating unfractionated heparin in this respect. The ability of mucopolysaccharides to interact with purified thrombospondin suggests a role for such molecules in the regulation of the biologic activity of thrombospondin, possibly in interactions with connective tissue, such as the subendothelium. Given that there are three binding sites per molecule and that thrombospondin appears to form polymeric aggregates with itself, significant binding energies could be developed.
A light microscopic technique based on alkaline phosphatase histochemistry was developed to specifically quantitate Type II cells in the intact rat lung. Lungs were fixed in 4% neutral-buffered formalin containing 0.25 M sucrose and embedded in glycol methacrylate. Two micron thick sections were mounted on glass microscope slides. Alkaline phosphatase activity was localized by using naphthol AS-BI phosphate as substrate in 0.125 M 2-amino-2-methyl-1-propanol buffer containing 0.625 mM MgCl2 (pH 8.9). Sections were counterstained with Harris hematoxylin. Type II cells were the only cell type in the alveolar region containing alkaline phosphatase activity, an observation that was confirmed by using electron microscopic histochemistry. By combining the alkaline phosphatase staining technique with standard morphometric procedures, the proliferative response to a single intratracheal dose of 10 mg silica was followed as a function of time. Type II cells were significantly increased at all time points examined. Twenty eight days following silica, Type II cells had increased to (252 +/- 16) X 10(6) cells per set of lungs compared to a control value of (141 +/- 32) X 16(6) cells. The method presented is a simple and rapid technique for examining Type II cell population kinetics.
Type II cell hyperplasia and hypertrophy were quantitated in the lungs of rats exposed to silica by using intratracheal injection. Hypertrophic type II cells were separated from normal type II cells by means of centrifugal elutriation of cells dispersed from the lungs by using protease. Type II cell hypertrophy was also quantitated, in situ, by measuring cell profile areas in lung sections. As a means of distinguishing between the hyperplastic and hypertrophic responses of type II cells we followed the progression of these two responses as a function of time and of dose. Seven, 14, and 28 days after a single intratracheal injection of silica (10 mg/rat), hypertrophic type II cells accounted for 33, 35, and 57% of the total type II cells recovered from the lungs. In contrast, hypertrophic type II cells accounted for less than 15% of the type II cells from control lungs. The appearance of hypertrophic type II cells was dose-related when examined 2 weeks after dosing; however, after only 1 week, the response of the type II cell appeared independent of dose. The hypertrophic response of type II cells could not be separated from the hyperplastic response according to either dose or time responses. These data support the hypothesis that in the lungs of silica-treated rats, the hypertrophic type II cell may be the proliferative type II cell. In addition, the hypertrophic type II cell appears to underlie the marked increases in surfactant levels seen in silica-treated lungs.
We utilized two assays for metabolic cooperation in vitro, one in which cells were grown in monolayer and one in which the cells formed three-dimensional structures in a collagen gel matrix. Both assays required one of the test cell pair to be resistant both to ouabain and to thioguanine (deficient in hypoxanthine-guanine phosphoribosyltransferase). Normal mammary gland cells, cells from preneoplastic mouse hyperplastic alveolar nodules, and mouse mammary tumor cells were metabolically linked in vitro to the drug-resistant tumor cells. Ouabain abrogated communication between tumor cells and normal mammary gland cells and between tumor cells and preneoplastic cells but had no effect on communication between tumor cells.
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Several investigators have reported the appearance of hypertrophic type II cells in the lungs of silica-treated rats. The purpose of this study was to isolate and characterize these hypertrophic type II cells. Lungs were digested with trypsin and the released cells were separated by using a flow gradient during centrifugal elutriation. Type II cells from control lungs were distributed in the flow gradient as a single population, whereas type II cells from the lungs of silica-treated rats had a bimodal distribution suggesting the presence of two distinct populations of type II cells; one of these populations appeared hypertrophic (type IIB) and the other appeared normal (type II cells; one of these populations appeared hypertrophic (type IIB) and the other appeared normal (type IIA). These two populations of type II cells from silica-treated rats differed significantly in cell size and their lamellar body content. The mean volume of type IIA and type IIB cells was 350 +/- 38 micron 3 and 523 +/- 29 micron 3, respectively. The mean number of lamellar bodies in type IIA and type IIB cells was 77 +/- 53 and 131 +/- 84 per cell, respectively. The mean volume of lamellar bodies was 0.39 +/- 0.09 micron 3 in type IIA cells and 0.66 +/- 0.10 micron 3 in type IIB cells. Type IIA cells were not significantly different from type II cells from the lungs of untreated rats. The distribution of type II cells from silica-treated lungs was such that 2 weeks after a single intratracheal injection of silica (10 mg/rat) type IIB cells accounted for 39.2 +/- 6.4% of the total type II cells recovered after centrifugal elutriation. The general morphological appearance of the isolated type IIA and type IIB cells was similar to that observed in type II cells isolated from untreated rats. These data indicate that hypertrophic type II cells may be isolated from the lungs of silica-treated rats and separated from normal type II cells thus allowing the role of these unusual type II cells in lung injury and repair to be investigated.
The ability of macrophages to induce drug-resistant variants was studied in an in vitro macrophage-tumor cell coculture system utilizing the hypoxanthine-guanine phosphoribosyl transferase locus as measured by resistance to 6-thioguanine. Tumor cells of mouse mammary tumor line 66 were sensitive to macrophage induction of thioguanine resistance as shown by an increase in the frequency of thioguanine-resistant variants which arose following macrophage coculture to levels at least 5- to 10-fold above the spontaneous frequency. Detection of increased numbers of variants depended upon the macrophage:tumor cell ratio, with 50:1 or greater being necessary. The activity of the macrophages was dependent upon their activation stage. The induction of drug-resistant variants could be inhibited by oxygen radical scavengers. The basis for the emergence of thioguanine-resistant cells appeared to be induction of new variants rather than selection of preexisting resistant cells from the parental population, since thioguanine-sensitive and -resistant cells were equally sensitive to macrophage-mediated toxicity. In six of the six macrophage-induced variants tested, resistance was associated with loss of hypoxanthine-guanine phosphoribosyl transferase activity. The reverse variation frequency at the hypoxanthine-guanine phosphoribosyl transferase locus in five macrophage-induced variants was low and similar to that of a stable ethyl methanesulfonate-induced, thioguanine-resistant line. Macrophages isolated directly from growing mammary tumors, as well as activated peritoneal macrophages, were capable of inducing thioguanine resistance in line 66 cells.
A series of subpopulation lines derived from a single mouse mammary tumor were tested for their ability to interact with each other in metabolic cooperation assays in three-dimensional collagen gel cultures. Inhibition of growth in the presence of selective drug (6-thioguanine or 2-fluoroadenine) of two 6-thioguanine-resistant cell lines, 66cl4 and 44FTO, as well as a 2-fluoroadenine-resistant line, 168FAR, occurred in the presence of other sensitive cells, demonstrating the transfer of drug sensitivity to drug-resistant cells. Transfer of drug resistance was shown by using selective medium containing hypoxanthine, aminopterin, and thymidine plus ouabain in which mutual metabolic cooperation between line 66cl4 (also ouabain resistant) and "wild-type" lines is required for growth. Effective communication leading to the transfer of drug resistance and ultimate survival of both cell types occurred in mixtures containing as little as 10% of either cell type. The requirement for mutual metabolic cooperation in hypoxanthine, aminopterin, and thymidine plus ouabain medium prevented line 66 from overgrowing line 66cl4 to the extent that it did in control medium. The ranked abilities of lines 66, 410.4, and 168 to promote growth when mixed with line 66cl4 in collagen cultures in hypoxanthine, aminopterin, and thymidine plus ouabain medium were correlated with the ranked cell densities required for these same cell lines to inhibit growth of line 66cl4 by metabolic cooperation in the presence of 6-thioguanine in monolayer cultures. These ranked abilities may be related to the effectiveness with which each of these cell lines is able to form communicating junctions with line 66cl4.
Macrophages were isolated by adherence from tumors produced by a number of murine mammary carcinoma lines and were examined by fluorescence-activated cell sorting for quantitation of leucine aminopeptidase and acid phosphatase. The tumors included three lines, 66, 67, and 168, which were originally derived from a single, spontaneously arising tumor in a BALB/cfC3H mouse and two other lines, D2A1 and D2F2, which were derived from a single tumor arising from the transplantable hyperplastic alveolar nodule line, D2. These five lines differ from one another in a number of characteristics, including the ability to metastasize spontaneously to the lung from subcutaneous implants and to form experimental metastases in lungs following intravenous injection. Line 67 is nonmetastatic under both circumstances, whereas lines 66, D2A1, and D2F2 are metastatic under the same conditions. Intermediate to these is line 168, which is nonmetastatic from the subcutaneous site but capable of colonizing the lung with an efficiency similar to 66 when injected IV. Tumor-associated macrophages (TAM) from lines 66, D2A1, and D2F2 contained the greatest amounts of leucine aminopeptidase (LAP) and those from line 67 the least, with TAM from 168 being intermediate. Conversely, the TAM from line 67 had the greatest amounts of acid phosphatase (APTase) and those from line 168 the least. In addition to differences among tumors in enzyme levels of the adherent TAM, the precentages of TAM that were adherent were also different among the tumors. Only 12% of TAM from line 67 were recovered in the adherent fraction as opposed to 35-38% of TAM from lines 66 and 168. These results confirm and extend our previous findings that TAM from metastatic tumors have increased levels of LAP compared to TAM from nonmetastatic tumors. They also demonstrate noncoordinate expression of LAP and APTase in TAM, and illustrate how a population of TAM can be homogeneous for one enzyme and heterogeneous for another. Furthermore, the difference in the percentage of macrophages that are adherent between metastatic and nonmetastatic tumors is another indication both of the heterogeneous nature of TAM and of the role of a tumor in determining the type of host infiltrate with which it is associated.