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Biomedical subjects

R E Miller

Publications and source records attributed to R E Miller.

At least 55 records · Page 3Linked to original sources
PubMed1997

Elements in the distal 5'-flanking sequence and the first intron function cooperatively to regulate glutamine synthetase transcription during adipocyte differentiation.

Glutamine synthetase (GS) expression increases dramatically during adipocyte differentiation of confluent 3T3-L1 cells. To identify differentiation-responsive cis-acting elements in the GS gene, several GSfusion genes were prepared and analyzed in stably transfected 3T3-L1 cells under conditions that trigger adipocyte differentiation. We find that the GS proximal 5'-flanking sequence lacks the regulatory elements required for differentiation-responsive expression. In contrast, a 2 kb intron 1 restriction fragment fused upstream of a heterologous promoter does drive reporter gene expression during hormone-triggered differentiation. The enhancer activity was localized to a 310 bp sequence near the middle of intron 1. Expression of fusion genes that include this 310 bp sequence does not temporally coincide with native gene expression. However, a composite gene that includes a far upstream GS sequence and the 2 kb intron 1 sequence yields a qualitatively different pattern of expression that closely resembles that of the native GS gene. The far upstream sequence alone exhibits no enhancer activity. Electrophoretic mobility shift analyses indicate that a 32 bp sequence within the 310 bp functional enhancer specifically binds differentiation-associated nuclear proteins. Although a C/EBP consensus sequence occurs in the 32 bp fragment, supershift analyses exclude C/EBP isoforms as the binding factor. In contrast, mutational analysis of the putative enhancer suggests that an HNF-3 isoform is involved. Thus our data indicate that elements in the distal 5'-flanking sequence and the first intron function cooperatively to regulate GS transcription and that HNF-3 may participate in that regulation.

3T3 Cells↗
PubMed1997

Flt3 ligand induces tumor regression and antitumor immune responses in vivo.

Daily treatment of mice with recombinant human Flt3 ligand (huFlt3L) results in a dramatic numerical increase in the number of dendritic cells (DCs) in vivo. Since DCs are pivotal in the induction of immune responses, we tested whether Flt3L treatment of mice challenged with a syngeneic methylcholanthrene (MCA)-induced fibrosarcoma would augment the generation of effective antitumor immune responses in vivo. Flt3L treatment not only induced complete tumor regression in a significant proportion of mice, but also decreased tumor growth rate in the remaining mice. A preliminary characterization of the cellular mechanisms involved suggests that Flt3L may be important in the treatment of cancer in situ through the generation of specific antitumor immune responses.

Animals↗
PubMed1997

High extracellular glucose impairs cardiac E-C coupling in a glycosylation-dependent manner.

Hyperglycemia is a major manifestation of all forms of diabetes mellitus and is associated with increased risk of cardiovascular disease. It is well established that cardiac excitation-contraction (E-C) coupling is adversely affected in diabetic animals such that ventricular myocyte action potential duration is prolonged and intracellular Ca2+ clearing and mechanical relaxation are slowed. We now report that ventricular myocytes incubated in a culture medium containing high extracellular glucose (25.5 mM) also exhibit these same changes in E-C coupling. These effects are not manifested for approximately 24 h after exposure. Furthermore, in the presence of normal glucose (5.5 mM), relaxation is also prolonged by fructose (20 mM), yet is unaffected by equimolar concentrations of nonmetabolizable sugars such as L-glucose and mannitol, implying that the high glucose effects require glucose entry into the cell and metabolic processing. The prolonged relaxation can also be produced by 5 mM glucosamine (an intermediate of glycosylation) and is blocked by 0.5 microgram/ml tunicamycin (an inhibitor of N-linked glycoprotein synthesis). Culturing myocytes with an inhibitor of glycation (10 mM aminoguanidine) does not prevent the high extracellular glucose concentration effects. Thus our data indicate that high extracellular glucose impairs cellular mechanisms contributing to myocardial relaxation and that this impairment may involve glycosylation of nascent proteins.

Action Potentials↗
PubMed1997

Presumptive red maple (Acer rubrum) toxicosis in Grevy's zebra (Equus grevyi).

Two female Grevy's zebras (Equus grevyi), one juvenile and one adult, were treated for hemolytic anemia. The juvenile survived, but the adult animal, which also had methemoglobinemia, was euthanized after it failed to recover from anesthesia. Significant pathologic findings in the adult zebra included generalized icterus, hemoglobinuric nephrosis, and paracentral hepatic necrosis. Serum titers for known infectious causes of anemia were negative. Examination of the zebra holding areas revealed two hybrid red maple (Acer sp.) trees. There was no known exposure to other hemolytic agents. This is the first report of probable red maple-induced hemolysis in zebra.

Anemia, Hemolytic↗
PubMed1997

Serial tranquilization of a reticulated giraffe (Giraffa camelopardalis reticulata) using xylazine.

A 5-yr-old captive-born reticulated giraffe (Giraffa camelopardalis reticulata) was tranquilized with i.m. xylazine 34 times in a 39-day period to allow nursing by her calf. Xylazine administered at approximately 0.16 mg/kg i.m. provided adequate sedation of the dam without detrimental side effects to the dam or the calf. While the dam was sedated, the calf was able to ingest adequate amounts of milk to produce acceptable weight gains until he was consistently eating solid foods.

Animals↗
PubMed1996

Impaired survival and proliferation in IL-7 receptor-deficient peripheral T cells.

Mice genetically deficient in IL-7R(alpha) are highly lymphopenic in the peripheral lymphoid organs. The functional competence of T cells that have developed in the absence of an IL-7R signal was investigated. Three important observations were made using several in vitro activation regimens. First, stimulation of T cells from IL-7R -/- mice at limiting dilution with immobilized Abs to CD3, CD4 or CD8, and CD18 revealed a six- to sevenfold reduction in the frequency of clonogenic T cells compared with T cells from IL-7R +/+ mice. IL-7R -/- T cells were also significantly less responsive to alloantigen as well as to receptor-independent stimuli such as PMA and ionomycin. Furthermore, the average clone size of single IL-7R -/- T cells was 50% smaller than that of IL-7R +/+ T cells. These data suggest that the reduced clonogenicity was predominantly due to intrinsic deficiencies in the ability of IL-7R -/- T cells to proliferate upon stimulation. Second, analysis of the kinetics of cell growth of IL-7R -/- T cells revealed that a significant proportion of T cells failed to proliferate within the first 72 h of in vitro stimulation, with the majority undergoing programmed cell death. Third, both clonogenic IL-7 -/- T cells and IL-7R +/+ T cells showed a similar proliferative response in the presence of IL-2 and similar survival kinetics, indicating that a subpopulation of IL-7R -/- T cells is functionally mature. We propose that an absence of IL-7R signaling not only affects T cell development in the thymus, but also results in the accumulation of functionally inactive T cells in the periphery.

Animals↗
PubMed1996

Structure-based design of parasitic protease inhibitors.

To streamline the preclinical phase of pharmaceutical development, we have explored the utility of structural data on the molecular target and synergy between computational and medicinal chemistry. We have concentrated on parasitic infectious diseases with a particular emphasis on the development of specific noncovalent inhibitors of proteases that play a key role in the parasites' life cycles. Frequently, the structure of the enzyme target of pharmaceutical interest is not available. In this setting we have modeled the structure of the relevant enzyme by virtue of its sequence similarity with proteins of known structure. For example, we have constructed a homology-based model of falcipain, the trophozoite cysteine protease, and used the computational ligand identification algorithm DOCK to identify in compuo enzyme inhibitors including oxalic bis(2-hydroxy-1-naphthyl-methylene)hydrazide (1) [Ring, C. S.; Sun, E.; McKerow, J. H.; Lee, G.; Rosenthal, P. J., Kuntz, I. D.; Cohen, F. E., Proc. Natl Acad. Sci. U.S.A. 1993, 90, 3583]. Compound 1 inhibits falcipain (IC50 6 microM) and the organism in vitro as judged by hypoxanthine uptake (IC50 7 microM). Following this lead, to date, we have identified potent bis arylacylhydrazides (IC50 150 nM) and chalcones (IC50 200 nM) that are active against both chloroquine-sensitive and chloroquine-resistant strains of malaria. In a second example, cruzain, the crystallographically determined structure of a papain-like cysteine protease, resolved to 2.35 A, was available. Aided by DOCK, we have identified a family of bis-arylacylhydrazides that are potent inhibitors of cruzain (IC50 600 microM). These compounds represent useful leads for pharmaceutical development over strict enzyme inhibition criteria in a structure-based design program.

Aldehydes↗
PubMed1995

In vitro antimalarial activity of chalcones and their derivatives.

A series of chalcones and their derivatives have been synthesized and identified as novel potential antimalarials using both molecular modeling and in vitro testing against the intact parasite. A large number of chalcones and their derivatives were prepared using one-step Claisen-Schmidt condensations of aldehydes with methyl ketones. These condensates were screened in vitro against both chloroquine-sensitive and chloroquine-resistant strains of Plasmodium falciparum and shown to be active at concentrations in the nanomolar range. The most active chalcone derivative, 1-(2,5-dichlorophenyl)-3-(4-quinolinyl)-2-propen-1-one (7), had an IC50 value of 200 nM against both a chloroquine-resistant strain (W2) and a chloroquine-sensitive strain (D6). The resistance indexes for all compounds were substantially lower than for chloroquine, suggesting that this series will be active against chloroquine-resistant malaria. Structure-activity relationships (SAR) of the chalcones in the context of a homology-based model structure of the malaria trophozoite cysteine protease, the most likely target enzyme, are presented.

Animals↗
PubMed1995

Induction of apoptosis in mature T cells by tumour necrosis factor.

T-cell receptor-induced apoptosis regulates immune responses and can result from interactions between Fas (Apo1/CD95) and Fas ligand (FasL). Mutations in the genes for Fas and FasL cause disorders resembling human autoimmune diseases in lpr and gld mice, respectively. However, peripheral T-cell deletion takes place in lpr mice, and autoimmune syndromes occur in mouse strains without Fas or FasL defects. Here we show that tumour necrosis factor (TNF) can mediate mature T-cell receptor-induced apoptosis through the p75 TNF receptor. Blockage of both TNF and FasL is required to abrogate T-cell death and TNF mediates the death of most CD8+ T cells, whereas FasL mediates the death of most CD4+ T cells. Our results suggest that autoregulatory apoptosis of the mature T cells can occur by two distinct molecular mechanisms.

Animals↗
PubMed1995

Antimalarial activity of new dihydroartemisinin derivatives. 6. alpha-Alkylbenzylic ethers.

A series of diastereomeric dihydroartemisinin alpha-alkylbenzylic ethers was synthesized in search for analogs with higher antimalarial efficacy and longer plasma half-life than the existing artemisinin derivatives. Artelinic acid was used as the model molecule for the design of new analogs. Two approaches were taken in an attempt to (a) increase the lipophilicity of the molecule and (b) decrease the rate of oxidative dealkylation of the target compounds. All compounds in this study showed at least equal or better in vitro antimalarial activity against Plasmodium falciparum than artelinic acid. The most active compounds of this series showed 10-, 20-, and 40-fold better inhibitory activity than artemether, artemisinin, and artelinic acid, respectively. Compounds which have a small methyl group substituted at the alpha-methylene group showed weaker activity than compounds with a larger carbethoxyalkyl substituent, indicating that the lipophilicity and the steric effect of the molecules play important roles in their antimalarial activity. This fact is further substantiated by the significantly weaker antimalarial activity of the carboxylic acids than their corresponding esters. Compounds with electron-withdrawing function (NO2) substantially increase the antimalarial activity. The S-diastereomers, in general, are severalfold more potent than the corresponding R-isomer.

Animals↗
PubMed1995

Inhibition of mature IL-1 beta production in murine macrophages and a murine model of inflammation by WIN 67694, an inhibitor of IL-1 beta converting enzyme.

The proinflammatory cytokine IL-1 beta is synthesized by activated monocytes and macrophages as a 31-kDa, biologically inactive precursor that is proteolytically processed to the biologically active 17-kDa mature molecule by the IL-1 beta converting enzyme (ICE). WIN 67694, Z-Val-Ala-Asp-CH2O(CO)[2,6-(CI2)]Ph, is a potent, selective inhibitor of human ICE. In activated murine peritoneal macrophages, WIN 67694 inhibited the release of mature IL-1 beta with an IC50 of 1.8 microM without any effect on the release of IL-1 alpha, IL-6, or TNF-alpha. The effect was specific to mature IL-1 beta release; the ICE inhibitor did not effect IL-1 beta RNA levels or precursor protein synthesis. In vivo, WIN 67694 was also able to inhibit selectively the release of IL-1 beta in a dose-dependent manner in a subcutaneous tissue chamber implant model of inflammation. IL-1 beta levels in tissue chamber fluid were inhibited 35 and 55% at 10 and 100 mg/kg, respectively. IL-1 alpha, IL-6, and TNF-alpha levels were not affected. The ability to selectively inhibit mature IL-1 beta release in vivo with ICE inhibitors will allow for detailed studies of the role of IL-1 beta and ICE in inflammatory diseases.

Amino Acid Sequence↗
PubMed1995

Management of common bile duct stones in the era of laparoscopic cholecystectomy.

PURPOSE: To assess the safety and efficacy of endoscopic retrograde cholangiopancreatography (ERCP) prior to laparoscopic cholecystectomy. METHODS: In patients suspected of harboring common duct stones, we performed ERCP prior to laparoscopic cholecystectomy (LC). Indications included jaundice, gallstone pancreatitis, elevated liver function tests, and visualizing a common duct stone and/or a dilated common duct on ultrasonography. Data were analyzed retrospectively. RESULTS: Of 217 patients undergoing LC, 37 (17%) had ERCP with or without endoscopic sphincterotomy (ES). Of these 37, common duct stones were noted in 19 patients (51%). Only 1 of 11 (9%) patients with mild gallstone pancreatitis had choledocholithiasis. The only complication following ERCP was pancreatitis in 1 patient who underwent uneventful LC. There were no deaths in the entire series. CONCLUSIONS: ERCP and ES is a safe and effective method of clearing the common duct of stones prior to LC. Patients with mild gallstone pancreatitis do not require ERCP prior to LC.

Adolescent↗