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Biomedical subjects

W J Muller

Publications and source records attributed to W J Muller.

89 records · Page 5Linked to original sources
PubMed1992

Induction of mammary tumors by expression of polyomavirus middle T oncogene: a transgenic mouse model for metastatic disease.

The effect of mammary gland-specific expression of the polyomavirus middle T antigen was examined by establishing lines of transgenic mice that carry the middle T oncogene under the transcriptional control of the mouse mammary tumor virus promoter/enhancer. By contrast to most transgenic strains carrying activated oncogenes, expression of polyomavirus middle T antigen resulted in the widespread transformation of the mammary epithelium and the rapid production of multifocal mammary adenocarcinomas. Interestingly, the majority of the tumor-bearing transgenic mice developed secondary metastatic tumors in the lung. Taken together, these results suggest that middle T antigen acts as a potent oncogene in the mammary epithelium and that cells that express it possess an enhanced metastatic potential.

Animals↗
PubMed1991

Expression of activated oncogenes in the murine mammary gland: transgenic models for human breast cancer.

Breast cancer is the leading cause of death among non-smoking women and thus has been the focus of intensive research. It has been generally accepted that the deregulation of oncogenes or their regulators play a pivotal role in progression of this prevalent disease. For example, amplification and overexpression of a number of oncogenes has been observed in a proportion of primary breast cancer biopsies. More recently, there has also been reports of inactivation tumor suppressor genes in human breast cancer. While there is compelling evidence for a role of these genes in breast cancer tumor progression due to limitations inherent in these studies it is difficult to establish a direct causal association between expression of a certain oncogene and tumor progression. For this reason many groups have employed the transgenic mouse as a model system to directly study effects of oncogene expression in the murine mammary gland. This review will attempt to highlight some of the important lessons and potential applications that have emerged from the study of oncogene expression in the mammary epithelium of transgenic mice. The utility of the transgenic system to assess the transforming potential of oncogenes, to investigate the multi-step nature of malignant progression, and to be used as models for therapeutic intervention will be discussed.

Animals↗
PubMed1990

The int-2 gene product acts as an epithelial growth factor in transgenic mice.

The induction of mammary tumors by mouse mammary tumor virus (MMTV) is thought to occur through proviral activation of one or more cellular genes. One of these, int-2, encodes a 27 kd protein which exhibits striking homology to the basic fibroblast growth factor family. To assess directly the role of the int-2 protein in cell proliferation, we have established transgenic mice which carry the int-2 gene driven by the MMTV promoter/enhancer. Expression of the int-2 gene in female transgenic mice results in pronounced mammary gland hyperplasia. Interestingly, expression of the MMTV-int-2 transgene in the prostate gland of male carriers results in a benign, but dramatic, epithelial hyperplasia similar to benign prostatic hypertrophy (BPH), a common but poorly understood disorder in human populations. Together, these results indicate that the int-2 product can act as a potent growth factor in these epithelial tissues.

Animals↗
PubMed1990

Studies of wall shear and mass transfer in a large scale model of neonatal high-frequency jet ventilation.

The problem of endotracheal erosion associated with neonatal high-frequency jet ventilation (HFJV) is investigated through measurement of air velocity profiles in a scaled up model of the system. Fluid mechanical scaling principles are applied in order to construct a model within which velocity profiles are measured by hot-wire anemometry. The effects of two different jet geometries are investigated. Velocity gradients measured near the tracheal wall are used to measure the shear stresses caused by the jet flow on the wall. The Chilton-Colburn analogy between the transport of momentum and mass is applied to investigate tracheal drying caused by the high shear flow. Shear forces are seen to be more than two times higher for jets located near the endotracheal tube wall than for those located axisymmetrically in the center of the tube. Since water vapor fluxes are dependent on these shears, they are also higher for the asymmetric case. Fluxes are shown to be greatly dependent on the temperature and relative humidity of the inspired gas. Water from the tracheal surface may be depleted within one second if inspired gases are inadequately heated and humidified. It is recommended that the design of neonatal HFJV devices include delivery of heated (near body temperature), humidified (as close to 100% humidity as possible) gases through an axisymmetric jet to best avoid the problem of endotracheal erosion.

High-Frequency Jet Ventilation↗
PubMed1990

A model of cigarette smoke particle deposition.

A computer model of aerosol deposition has been extended to cover particle sizes representative of cigarette mainstream and sidestream smoke particles. The model is the first to theoretically predict total airway depositions of mainstream particles in a range which agrees with experimentally determined literature values by including effects of hygroscopicity and normal smoking breathing patterns. The hygroscopic characteristics of cigarette smoke particles are modeled as if they were saturated sodium chloride droplets. A discussion is included showing that this assumption is consistent with presently available data on the hygroscopic characteristics of cigarette smoke. Detailed regional depositions are provided. Though most of the particles are shown to deposit in the periphery, the surface concentrations of deposited particles are not necessarily much greater there than in centrally located airways. A peak in surface concentration at the third generation is exhibited, despite low total depositions there. Central airway surface concentrations are shown to be relatively independent of breathing pattern and airway geometry, implying that the effects of cigarette smoke particle deposition cannot be greatly reduced by changing the pattern of smoke inhalation. For sidestream smoke particles, total percent depositions agree with literature values of 7%-20% for both nonhygroscopic and hygroscopic particles. Deposition is seen to be favored in the periphery of the lung, though surface concentrations of the deposited material can be greater in Weibel Generations 3-6. Peak surface concentrations are again seen to occur in Generation 3. The increased toxicity of sidestream smoke particles may make them as unhealthy as mainstream smoke particles, despite the higher depositions observed for mainstream smoke.

Computer Simulation↗
PubMed1989

Convective mixing mechanisms in high frequency intermittent jet ventilation.

A liquid flow visualization technique was used to identify the location of neutrally buoyant bead clouds injected into airway models during flows simulating high frequency intermittent jet ventilation (HFIJV) in neonatal lungs. The motions of these bead clouds show that the convective or bulk mixing that occurs during HFIJV is made up of two parts; a turbulent convective exchange with the atmosphere caused by the jet in the trachea and a streaming motion along the airways driven by an interaction between the jet and the expansion and contraction of the airways due to their compliance. These convective streaming motions combine with molecular diffusion to produce augmented diffusion which transports O2 and CO2 between the trachea and the peripheral alveoli. Optimizing HFIJV (as well as other forms of HFV) depends on maximizing these airway convective streaming flows which depend on many more lung and fluid mechanical parameters than are necessary to describe conventional mechanical ventilation.

High-Frequency Jet Ventilation↗
PubMed1988

Single-step induction of mammary adenocarcinoma in transgenic mice bearing the activated c-neu oncogene.

We have used transgenic mice that carry an activated c-neu oncogene driven by a mouse mammary tumor virus (MMTV) promoter to assess the stepwise progression of carcinogenesis in mammary epithelium. Unlike the stochastic occurrence of solitary mammary tumors in transgenic mice bearing the MMTV/c-myc or the MMTV/v-Ha-ras oncogenes, transgenic mice uniformly expressing the MMTV/c-neu gene develop mammary adenocarcinomas that involve the entire epithelium in each gland. Because these tumors arise synchronously and are polyclonal in origin, expression of the activated c-neu oncogene appears to be sufficient to induce malignant transformation in this tissue in a single step. In contrast, expression of the c-neu transgene in the parotid gland or epididymis leads to benign, bilateral epithelial hypertrophy and hyperplasia which does not progress to full malignant transformation during the observation period. These results indicate that the combination of activated oncogene and tissue context are major determinants of malignant progression and that expression of the activated form of c-neu in the mammary epithelium has particularly deleterious consequences.

Adenocarcinoma↗
PubMed1988

The polyomavirus enhancer comprises multiple functional elements.

The polyomavirus enhancer occupies 244 base pairs within noncoding sequences between the early and late transcription units. To define more precisely the DNA sequences that make up the enhancer, we cloned it together with the viral early promoter upstream of a reporter gene, isolated mutants bearing deletions introduced in vitro in the enhancer, and measured the capacity of the various mutant genomes to express the cat gene after transient transfection into mouse 3T3 cells. Analysis of a large number of deletion mutants revealed that the enhancer is between 102 and 172 base pairs long and can be divided into at least three functional elements. Relative to the entire enhancer, individual elements possessed little or no enhancer activity. However, pairs of elements enhanced transcription to levels much higher than the sum of individual elements approximating the activity of the complete enhancer. These findings support the view that the polyomavirus enhancer is composed of multiple sequence elements that function combinatorily and imply that a measure of cooperation exists in the interaction between cellular protein factors bound to their cognate sites in the enhancer and the transcriptional machinery of the cell.

Animals↗
PubMed1988

Multiple subelements within the polyomavirus enhancer function synergistically to activate DNA replication.

The polyomavirus origin for DNA replication comprises at least two essential, but functionally distinct, cis-acting components. One of these, the origin core, is required only for DNA replication. It includes binding sites for large T antigen and the origin of bidirectional DNA replication. The other component is required for both transcription and DNA replication and is represented by two functionally redundant regions, alpha and beta, which are elements of the polyomavirus enhancer. Whereas either enhancer element will activate DNA replication, both enhancer elements are required to constitute a functional enhancer of transcription. To identify the sequences that make up each enhancer element, we have subjected them separately to in vitro mutagenesis and measured their capacity to activate replication in cis of the origin core in MOP-8 cells, which provide all trans-acting replicative functions including large T antigen. The results reveal that the beta enhancer element is composed of three subelements, two auxiliary subelements, and a core subelement. The core subelement independently activated DNA replication, albeit poorly. The auxiliary subelements, which were inactive on their own, acted synergistically with the core subelement to increase its activity. Interestingly, dimers of the beta core subelement functioned as well as the combination of a beta auxiliary subelement and a core subelement, suggesting that the subelements are functionally equivalent. The alpha enhancer element is organized similarly; it too comprises an auxiliary subelement and a core subelement. These results lead us to suggest that the polyomavirus enhancer comprises two levels of organization; two or more enhancer elements form an enhancer, and two or more subelements make up an enhancer element. The subelements share few sequences and serve as binding sites for distinct cellular factors. It appears, therefore, that a number of different cellular proteins function cooperatively to activate polyomavirus DNA replication by a common mechanism.

Base Sequence↗
PubMed1987

Airway deposition of hygroscopic heterodispersed aerosols: results of a computer calculation.

A new computer model is developed and used to calculate the deposition of inhaled heterodispersed hygroscopic aerosols for mouth breathing in a Weibel symmetric bronchial tree. The model was first validated by obtaining good agreement with recent experimental and theoretical data on regional and total airway deposition of monodispersed and heterodispersed nonhygroscopic aerosols. The model was then used to obtain predictions of regional and total deposition of heterodispersed hygroscopic aerosol particles (droplets of NaCl solutions). Parameters that were varied in the hygroscopic calculations include initial droplet NaCl concentration, time of inspiration and expiration, volume of aerosol inspired, period of breath holding, and initial inhaled lognormal aerosol mass median diameter and geometric standard deviation. Results of the computer calculations show that increasing heterodispersity tends to flatten and broaden regional deposition curves when fraction of inhaled mass deposited is plotted vs. inhaled mass median aerodynamic particle diameter. Hygroscopicity is shown to increase tracheobronchial and pulmonary airway deposition with hypertonic NaCl solution aerosols showing increases over isotonic and nonhygroscopic aerosols of up to 200%.

Aerosols↗
PubMed1985

Sequence-dependent DNA replication in preimplantation mouse embryos.

Circular, double-stranded DNA molecules were injected into nuclei of mouse oocytes and one- or two-cell embryos to determine whether specific sequences were required to replicate DNA during mouse development. Although all of the injected DNAs were stable, replication of plasmid pML-1 DNA was not detected unless it contained either polyomavirus (PyV) or simian virus 40 (SV40) DNA sequences. Replication occurred in embryos, but not in oocytes. PyV DNA, either alone or recombined with pML-1, underwent multiple rounds of replication to produce superhelical and relaxed circular monomers after injection into one- or two-cell embryos. SV40 DNA also replicated, but only 3% as well as PyV DNA. Coinjection of PyV DNA with either pML-1 or SV40 had no effect on the replicating properties of the three DNAs. These results are consistent with a requirement for specific cis-acting sequences to replicate DNA in mammalian embryos, in contrast to sequence-independent replication of DNA injected into Xenopus eggs. Furthermore, PyV DNA replication in mouse embryos required PyV large T-antigen and either the alpha-beta-core or beta-core configuration of the PyV origin of replication. Although the alpha-core configuration replicated in differentiated mouse cells, it failed to replicate in mouse embryos, demonstrating cell-specific activation of an origin of replication. Replication or expression of PyV DNA interfered with normal embryonic development. These results reveal that mouse embryos are permissive for PyV DNA replication, in contrast to the absence of PyV DNA replication and gene expression in mouse embryonal carcinoma cells.

Animals↗
PubMed1984

Isolation of large T antigen-producing mouse cell lines capable of supporting replication of polyomavirus-plasmid recombinants.

Construction of polyomavirus vectors, analysis of mutant viruses, and rescue of integrated polyomavirus genomes would be considerably aided by the availability of transformed, permissive mouse cell lines capable of producing the viral tumor antigens. To isolate such cell lines, we constructed a hybrid transcription unit composed of the simian virus 40 early promoter fused to the coding region for the polyomavirus tumor antigens. This hybrid transcription unit was used to transform NIH 3T3 cells. Independent foci of transformed cells were isolated, recloned, and characterized. Among 10 lines initially analyzed, 7 supported the replication of origin-bearing plasmid DNAs. Three cell lines were characterized in greater detail. Each line contained one or two independent insertions of polyomavirus DNA and synthesized all three viral tumor antigens. Moreover, the large tumor antigen in two of three lines bound with specificity to sequences about the polyomavirus origin and early promoter. These cell lines should prove useful for studying not only the replication of polyomavirus but also the expression of foreign genes in a mouse cell environment.

Animals↗
PubMed1983

Polyomavirus origin for DNA replication comprises multiple genetic elements.

To define the minimal cis-acting sequences required for polyomavirus DNA replication (ori), we constructed a number of polyomavirus-plasmid recombinants and measured their replicative capacity after transfection of a permissive mouse cell line capable of providing polyomavirus large T antigen in trans (MOP cells). Recombinant plasmids containing a 251-base-pair fragment of noncoding viral DNA replicate efficiently in MOP cells. Mutational analyses of these viral sequences revealed that they can be physically separated into two genetic elements. One of these elements, termed the core, contains an adenine-thymine-rich area, a 32-base-pair guanine-cytosine-rich palindrome, and a large T antigen binding site, and likely includes the site from which bidirectional DNA replication initiates. The other, termed beta, is located adjacent to the core near the late region and is devoid of outstanding sequence features. Surprisingly, another sequence element named alpha, located adjacent to beta but outside the borders of the 251-base-pair fragment, can functionally substitute for beta. This sequence too contains no readily recognized sequence features and possesses no obvious homology to the beta element. The three elements together occupy a contiguous noncoding stretch of DNA no more than 345 base pairs in length in the order alpha, beta, and core. These results indicate that the polyomavirus origin for DNA replication comprises multiple genetic elements.

Animals↗
PubMed1983

Polyomavirus-plasmid recombinants capable of replicating have an enhanced transforming potential.

The frequency of transformation of rodent fibroblasts by polyomavirus is enhanced by a viral gene product, large T-antigen. However, this effect of large T-antigen cannot be demonstrated with pBR322-cloned viral DNA. Recently, it was discovered that pBR322 contains cis-acting sequences inhibitory to DNA replication in mammalian cells. Because polyomavirus large T-antigen is required for viral DNA replication, we examined the possibility that our inability to demonstrate a requirement for large T-antigen in transformation with pBR322-cloned viral DNA was due to the failure of the chimeric DNA to replicate in the transfected cells. To this end we constructed polyomavirus recombinant molecules with a plasmid (pML-2) that lacks these "poison" sequences and measured their capacity to transform cells. Here we report that recombinant plasmids capable of replicating in the transfected cells transform these cells at frequencies approximately sixfold greater than their replication-defective counterparts.

Animals↗
PubMedDate not supplied

A p75 tumor necrosis factor receptor-specific mutant of murine tumor necrosis factor alpha expressed from an adenovirus vector induces an antitumor response with reduced toxicity.

The toxic effects of tumor necrosis factor alpha (TNFalpha) have greatly limited its use in tumor therapy. Recently, clear evidence has been obtained linking the p55 TNF receptor (TNFR) to the induction of systemic toxicity. We have generated a p75 murine TNFR (mTNFR)-specific mutant of mTNFalpha (D142N-A144R), cloned this gene into a recombinant adenovirus vector (Ad-75), and studied its efficacy for tumor immunotherapy of a murine transgenic breast cancer model. Cell culture supernatants from Ad-75-transduced cells showed no cytotoxic activity on L929 cells, but retained the ability to induce proliferation of a murine T-cell line (CT6); this activity was not blocked by soluble p55 mTNFR. Furthermore, it was shown that the mutant form of mTNFalpha was able to coimmunoprecipitate only with the p75 mTNFR and not with the p55 mTNFR. Tumors injected with Ad-75 became necrotic, and mice injected with < or =1 x 10(9) plaque-forming units showed no mortality, whereas both wild-type murine and human TNF vectors induced lethality at doses of 1 and 5 x 10(8) plaque-forming units. All Ad-TNF vectors induced partial or permanent tumor regressions, with cured mice showing immune memory against the tumor. These results demonstrate that a p75 mTNFR agonist expressed from a recombinant adenovirus vector does not induce mortality at doses that cause tumor regression.

Adenoviridae↗