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J A Sawicki

Publications and source records attributed to J A Sawicki.

17 recordsLinked to original sources
PubMed2000

K6/ODC transgenic mice as a sensitive model for carcinogen identification.

Ornithine decarboxylase (ODC), an important enzyme in the polyamine biosynthetic pathway, is aberrantly regulated in many epithelial tumors of rodents and humans. In murine skin, it has been shown that ODC overexpression provides a sufficient condition for tumor promotion. Therefore, we hypothesized that K6/ODC transgenic mice in which ODC overexpression was targeted to hair follicle keratinocytes might provide a sensitive model for identifying genotoxic carcinogens. Ten known carcinogens or noncarcinogens have been tested in the model so far and results are highly concordant with 2-year rodent bioassays (100% concordant). More importantly, each of two chemicals tested that is recognized as a human carcinogen was identified as a carcinogen in K6/ODC transgenic mice. In addition, 7, 12-dimethylbenz(a)anthracene (DMBA) dose response studies indicated that even at a very low dose, 2 nmol, a high percentage of mice (50%) had already developed tumors 8 weeks after treatment. We conclude that the K6/ODC transgenic mouse model is very sensitive to topical application of genotoxic carcinogens and could therefore be a useful mouse model for carcinogen identification and chemical risk assessment.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1998

A composite CMV-IE enhancer/beta-actin promoter is ubiquitously expressed in mouse cutaneous epithelium.

In most existing transgenic mouse models developed for the study of specific genes in the skin, the goal has been to target transgene expression to defined populations of cells in the cutaneous epithelium. Keratin promoters have been especially useful for this purpose. In some instances, however, it may be desirable to express a transgene in all the cells of the cutaneous epithelium. Since no ubiquitously expressed promoter sequences had previously been identified, we used lacZ reporter transgenes to test two enhancer/promoter sequences for ubiquitous expression in the skin of adult transgenic mice. We find that a CMV enhancer/CMV promoter is not active in most cell types in the skin, whereas a CMV enhancer/modified beta-actin promoter sequence is active in the suprabasal and basal cells of the epidermis as well as in the epithelial cells of the hair follicles, sebaceous glands, and the dermal papillae.

Actins↗
PubMed1998

Cell-specific ecdysone-inducible expression of FLP recombinase in mammalian cells.

The ability of site-specific recombinases, like FLP and Cre, to catalyze alterations in genomic DNA is well established, whereas their application to genetic engineering strategies has been restricted because of the inability to temporally regulate their expression and subsequent recombination events in specific populations of cells. We describe a regulatory system for ecdysone-controlled expression of FLP recombinase. Furthermore, we demonstrate that ecdysone-induced, FLP-mediated site-specific recombination events can be targeted to specific cells. This system can be applied to cell-lineage studies as well as to the design of gene-therapy strategies, particularly in stem cells.

3T3 Cells↗
PubMed1996

Elements in the murine c-mos messenger RNA 5'-untranslated region repress translation of downstream coding sequences.

Murine c-mos transcripts isolated from testes have 5'-untranslated regions (5'UTRs) of approximately 300 nucleotides with a series of four overlapping open reading frames (ORFs) upstream of the AUG codon that initiates the Mos ORF. Ovarian c-mos transcripts have shorter 5'UTRs (70-80 nucleotides) and contain only 1-2 of the upstream ORFs (uORFs). To test whether these 5'UTRs affect translational efficiency, we have constructed plasmids for the expression of chimeric transcripts with a mos-derived 5'UTR fused to the Escherichia coli beta-galactosidase coding region. Translational efficiency has been evaluated by measuring beta-galactosidase activity NIH3T3 cells transiently transfected with these plasmids and with plasmids where various mutations have been introduced into the 5'UTR. We show that the 5'UTR characteristic of testis-specific c-mos mRNA strongly represses translation relative to the translation of transcripts that contain a 5'UTR derived from beta-globin mRNA, and this is mainly due to the four uORFs. Each of the four upstream AUG triplets can be recognized as a start site for translation, and no single uAUG dominates the repressive effect. The uORFs repress translation by a mechanism that is not affected by the amino acid sequence in the COOH-terminal region of the uORF-encoded peptides. The very short uORF (AUGUGA) present in ovary-specific transcripts does not repress translation. Staining of testis sections from transgenic mice carrying chimeric beta-galactosidase transgene constructs, which contain a mos 5'UTR with or without the uATGs, suggests that the uORFs can dramatically change the pattern of expression in spermatogenic cells.

Animals↗
PubMed1995

Increased frequency of spontaneous skin tumors in transgenic mice which overexpress ornithine decarboxylase.

Ornithine decarboxylase, a critical regulatory enzyme for polyamine biosynthesis, is highly inducible by growth-promoting stimuli in mouse epidermis but the enzyme level is only transiently elevated due to rapid turnover of the protein. Here we report that constitutive overexpression of the enzyme in the skin of transgenic mice causes several phenotypic abnormalities. Effects observed include development of dermal follicular cysts, excessive skin wrinkling, enhanced nail growth, alopecia, and spontaneous tumor development. These results indicate that up-regulation of polyamine biosynthesis can profoundly disturb skin homeostasis and alter susceptibility to neoplastic development.

Animals↗
PubMed1991

Effects of exogenous putrescine on murine preimplantation development in vitro.

After first demonstrating that murine embryos take up putrescine from the medium in which they are cultured in vitro, fertilized eggs were placed in culture and maintained for 4 days in medium supplemented with varying amounts of putrescine. Their development was monitored each day. While embryos that were cultured in putrescine-supplemented medium developed at the same rate as control embryos, a significantly higher percentage of the putrescine-treated embryos attained the blastocyst stage as compared to the control group.

Animals↗
PubMed1986

Cellular protein differences between nontumorigenic Ad5 and tumorigenic Ad12 transformed mouse cells.

Murine fibroblasts transformed by adenovirus 12 (Ad12) show reduced class I major histocompatibility complex (MHC) antigen synthesis and form tumors in syngeneic mice whereas those transformed by adenovirus 5 (Ad5) show no alteration in class I antigen synthesis and do not form tumors (K. B. Eager, J. Williams, D. Breiding, S. Pan, B. Knowles, E. Appella, and R. P. Ricciardi (1985) Proc. Natl. Acad. Sci. USA 82, 5525-5529). Nearly 1500 metabolically labeled polypeptides from the Ad5 and Ad12 transformed cell lines as well as polypeptides from a nontransformed murine line of the same haplotype were compared by two-dimensional gel electrophoresis. In addition to the reduction of the class I H-2 transplantation antigens seen in the Ad12-transformed lines, we detect few but reproducible polypeptide differences between the tumorigenic and nontumorigenic cell lines.

Adenoviridae↗
PubMed1985

Identical RT1 class II molecules are expressed by rat RT1m and RT1c haplotypes.

The RT1m haplotype of MNR rats has been suggested to be a recombinant RT1 haplotype inheriting RT1.A (class I) alleles from RT1a (DA) and RT1.B (class II) alleles from RT1c (AUG). Additional serologic and biochemical assays, however, have suggested that RT1m and RT1c share a single identical RT1.B molecule, although differing in the expression of the second RT1.B molecule. To resolve this contradiction, RT1.B class II molecules, comparable to I-A and I-E molecules in mice, expressed by the RT1c and RT1m haplotypes were immunoprecipitated by cross-reactive mouse anti-Ia antibodies and were compared by two-dimensional gel electrophoresis and by high pressure liquid chromatographic separation of tryptic peptides. Respective subunits expressed by the two haplotypes co-migrate on two-dimensional gels and have identical tryptic peptide maps. The results at the protein level were confirmed at the DNA level by Southern blot analysis of MNR and AUG genomic DNA. Identical restriction fragments associated with the RT1m and RT1c haplotypes hybridized with each of the DC1 beta, DR alpha, and DR beta cDNA probes. The results at both the protein and DNA levels suggest that the RT1m and RT1c haplotypes share identical expressed alleles at the RT1.Ba, RT1.Bb, RT1.Bc, and RT1.Bd loci.

Animals↗
PubMed1984

Evidence for extensive polymorphism of RT1 Class II molecules in the rat.

RT1.B Class II molecules, comparable to I-A and I-E molecules in mice, have been characterized by two-dimensional (2D) gel electrophoresis for seven rat strains expressing different RT1 haplotypes. RT1.B molecules were immunoprecipitated from radiolabeled rat lymphocyte preparations with mouse monoclonal antibodies reactive with mouse I-Ab antigens and I-Ek antigens. For each RT1 haplotype, two I-A-like subunits, alpha and beta, and two I-E-like subunits, gamma and delta, have been identified. 2D gel analysis of RT1.B molecules from different strains in paired combinations reveals that, with a few notable exceptions, the alpha, beta, and delta subunits associated with different RT1 haplotypes are characterized by unique 2D gel spot patterns. In contrast, the gamma subunits of all RT1.B haplotypes appear identical. This evidence confirms and extends our previous analysis of RT1.B molecules by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and suggests a high level of polymorphism for genes in the RT1.B region, with the exception of the RT1.Bc gene encoding gamma. Interestingly, three rat strains appear exceptional in the identity of their alpha, gamma, and delta subunits, which suggests that these strains express closely related RT1 haplotypes derived from a common progenitor.

Animals↗
PubMed1982

Production of viable adult trisomy 17 reversible diploid mouse chimeras.

Aneuploid mouse embryos and fetuses are an important system for investigating the pathogenesis of the developmental and functional consequences of chromosome imbalance in mammals. However, the fact that almost all mouse aneuploids die in embryonic or fetal life restricts their usefulness for studies of loci and functions that are expressed only after birth. As an approach to rescuing aneuploid cells, we have prepared chimeras by aggregating aneuploid embryos with diploid embryos at the 8-16 cell stage. This technique has allowed us to produce trisomy 17 reversible diploid (Ts17 reversible 2n) chimeras containing cells of a trisomic state, which is ordinarily lethal at 10-12 days of gestation. All of the analyzed organs from the chimeras, including brain, liver, kidney, lung, muscle, heart, thymus, spleen, bone marrow, blood, and skin, contained both Ts17 and 2n cells. The proportion of Ts17 cells in each organ as estimated from coat color, enzyme markers (glucosephosphate isomerase), and karyotypes ranged from 5% to 85%, most commonly 20-40%. This is in contrast to the control 2n reversible 2n chimeras in which the 2n component on the same genetic background as the Ts17 generally comprised 60-90% of the cell population. The growth rates of the living Ts17 reversible 2n chimeras were in the lower half of the normal range, and the oldest animals are now age 14 mo. No progeny were obtained from Ts17 germ cells in the two fertile T217 reversible 2n chimeras. In comparison with analogous human situations, it is striking that, except for a kink in the tail of one living animal, none of the Ts17 reversible 2n chimeras had any discernible structural abnormalities. Our findings indicate that it is feasible to rescue cells from autosomal aneuploidies that otherwise result in early fetal death.

Aneuploidy↗
PubMed1982

Assignment of the gene for beta 2-microglobulin (B2m) to mouse chromosome 2.

We have assigned the gene (B2m) coding for murine beta 2-microglobulin (B2M) to mouse chromosome 2 by using a novel panel of Chinese hamster-mouse somatic cell hybrid clones. Because of 35 independent primary hybrids used in this study were derived from two types of feral mice, each with a different combination of Robertsonian translocation chromosomes, as well as from mice with a normal complement of acrocentric chromosomes, analysis of 16 selected mouse enzyme markers provided data on the segregation of all 20 mouse chromosomes in these hybrids. Mouse B2M was identified in cell hybrids by immunoprecipitation with a species-specific anti-mouse B2M antiserum followed by two-dimensional polyacrylamide gel electrophoresis of the immunoprecipitated polypeptides. Enzyme analysis of the segregant clones excluded all chromosomes for B2m assignment except mouse chromosome 2, and karyotype analysis of nine informative hybrid clones confirmed the assignment of B2m to this chromosome. These results demonstrate that, in the mouse, as in man, B2m is not linked to the major histocompatibility or immunoglobulin loci.

Animals↗
PubMed1981

H-2 antigens as genetic markers: a two-dimensional gel electrophoretic study.

Direct immunoprecipitation and two-dimensional (2D) gel electrophoresis have been used to identify and characterize genetic variation of the H-2K and H-2D regions. Using inbred strains of mice and alloantisera, haplotype-specific polypeptides were defined for five different H-2 haplotypes. Specific immunoprecipitates prepared from strains of different haplotypes were applied to 2D gels in pairwise combinations to determine whether peptides specific to one haplotype can be distinguished from peptides specific to another. Those haplotype-specific peptides that migrate to unique positions on 2D gels with respect to the positions occupied by haplotype-specific peptides of another haplotype are useful as biochemical genetic markers. Cross-reactivity among K- and D-region antigens of different haplotypes was identified on 2D gels and found to correlate well with existing data based on serological cross-reactivity. An anti-mouse beta 2-microglobulin serum was found to be a useful general reagent for immunoprecipitating haplotype-specific H-2 antigens to permit their visualization on 2D gels.

Animals↗
PubMedDate not supplied

Sibling rivalry and the new baby: anticipatory guidance and management strategies.

Sibling rivalry can be found in many families and frequently creates a stressful and challenging situation for parents. The arrival of a new baby often causes older siblings to feel displaced, frustrated, angry, and even unloved. Age, gender, personality and temperament, and parental behavior are factors that appear to influence the degree to which sibling rivalry occurs. Common reactions of older siblings to the birth of a new baby include aggression toward the newborn, behavioral regression, and attention seeking behavior, as well as independence and maturity. Anticipatory guidance is recommended to help parents adequately prepare their older child for the arrival of a new sibling. Strategies for managing sibling rivalry include open parent-child communication, equal treatment of siblings, non-intervention in sibling conflicts, distraction, and separation. Parents can minimize feelings of jealousy between siblings by providing a supportive, nurturing environment that allows each child to feel secure and loved.

Adaptation, Psychological↗