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J Fargnoli

Publications and source records attributed to J Fargnoli.

29 records · Page 2Linked to original sources
PubMed1991

Concomitant decline in heat-induced hyperthermia and HSP70 mRNA expression in aged rats.

An age-related impairment in the induction of heat-shock proteins (HSPs), which are thought to comprise a protective or adaptive response to cellular stress, may contribute to a reduction in thermal tolerance with age. When 5- and 24-mo-old rats were exposed to ambient temperatures of 23, 35, 37.5, or 40 degrees C for 90 min, a graded increase in the level of HSP70 mRNA expression was observed in brain, lung, and skin of animals from both groups. However, at temperatures above 23 degrees C, HSP70 expression was less in tissues of older rats when compared with those of young animals. This relative decline of heat-induced HSP70 mRNA levels with age correlated with an attenuated increase in colonic temperature (Tc) of old rats resulting from heat exposure. In other experiments, it was determined that the duration of hyperthermia was also an important factor in determining the level of HSP70 mRNA expression in vivo. Thus age-related differences in heat-induced HSP70 mRNA expression appear to result from differences in Tc due to heat stress rather than an impairment in the regulation of HSP70 gene expression.

Aging↗
PubMed1991

Progressive changes from young adult age to senescence in mRNA for rat cardiac myosin heavy chain genes.

In rodents of an advanced age the cardiac contraction exhibits a reduced shortening velocity and prolonged time course. This is, in part, due to a reduction in myosin ATPase activity which results from a shift in the myosin heavy chains from the alpha (V1) to the beta (V3) isoform. The present study demonstrates that during adult aging the steady state levels of alpha myosin heavy chain mRNA decrease while those of beta myosin heavy chain increase; for both mRNAs the respective changes are greater than fourfold. Thus, the phenotypic, biophysical and biochemical cardiac contractile changes with adult aging are, at least, in part regulated at the level of gene expression.

Aging↗
PubMed1990

Isolation and characterization of the hamster gadd153 gene. Activation of promoter activity by agents that damage DNA.

A group of five cDNA clones, representing the gadd genes, were recently isolated from Chinese hamster ovary (CHO) cells as genes induced upon growth arrest and after DNA damage (Fornace, A. J., Jr., Nebert, D. W., Hollander, M. C., Luethy, J. D., Papathanasiou, M., Fargnoli, J., and Holbrook, N. J. (1989) Mol. Cell. Biol. 9, 4196-4203). We have isolated and characterized one of these genes, gadd153. The gene spans five kilobases and contains four exons. The 5'-flanking region of the gene, within 420 base pairs of the transcription initiation site, contains a number of cis elements associated with transcriptional regulation in other genes. These include a Hogness box, ATAAAA, an inverted GCCAAT box; seven SP1 transcription factor binding sites, and an AP-1 site. This region is rich in G + C content (greater than 70%) and contains an unusually long stretch of alternating CpG residues. The 800-base pair region immediately upstream of the transcription start site can drive expression of the bacterial chloramphenicol acetyltransferase (CAT) gene, but only in its endogenous orientation, in three different cell lines: HeLa, CHO, and Jurkat. The gadd153 promoter is strongly activated by methyl methanesulfonate, hydrogen peroxide, and UV irradiation, but not by growth arrest signals. This suggests that separate and very different regulatory pathways are involved in the induction of the gadd153 gene by growth cessation and DNA damage.

Animals↗
PubMed1990

Discordant expression of heat shock protein mRNAs in tissues of heat-stressed rats.

Although the induction of heat shock proteins (HSP) has been studied extensively in cultured cells, comparatively few studies have examined their expression in vivo. In this report, mRNA expression of two HSP families, HSP70 and HSP27, was investigated in brain, liver, lung, and skin of rats exposed to elevated ambient temperatures. The time course and relative magnitude of the heat-induced expression for these two HSP differed between tissues of the same animal. Even within the same tissue, HSP70 and HSP27 displayed differential kinetics of induction. In brain, lung, and skin, induction of HSP70 was dependent on the duration and temperature of the heat stress. This induction was transient with maximal HSP70 expression occurring at 1 h and returning to baseline 3 h after removal of the animals from heat stress. In liver, HSP70 expression did not show a direct relationship with temperature conditions and maximal induction did not occur until 6 h after heat stress. Heat-induced HSP27 expression was dependent on time and temperature of exposure in lung and skin but not in brain and liver. These findings demonstrate that the heat shock response in vivo lacks much of the coordinate control of expression characteristic of cultured cell populations and suggest that mechanisms controlling this cellular stress response are influenced by physiologic factors that cannot be studied in vitro.

Animals↗
PubMed1990

Low-ratio hybridization subtraction.

A hybridization subtraction protocol that uses low ratios of RNA to cDNA has been developed to enrich for the cDNA of transcripts that are elevated in one cell population relative to another. This low-ratio hybridization subtraction protocol was found to yield substantial enrichment for the cDNA of low-abundance transcripts induced or increased only several fold. Conditions for the cloning of cDNA enriched by our hybridization subtraction and identification of clones coding for induced transcripts are presented. By screening the cDNA library with probes synthesized from the starting cDNA and cDNA enriched by low-ratio hybridization subtraction, clones coding for induced transcripts could be efficiently identified. The choice of reverse transcriptase used to synthesize the cDNA was found to be important for the enrichment of cDNA for longer length RNA. Low-ratio hybridization subtraction of cDNA synthesized with MMLV reverse transcriptase was effective for the enrichment of cDNA coding for RNA to at least 5 kb in length, while the AMV enzyme was effective only for the cDNA of shorter RNA (less than 1 kb). The characterization of several different low-ratio hybridization subtraction libraries is presented, and the advantages and disadvantages of various hybridization subtraction strategies are discussed.

Animals↗
PubMed1990

Decreased expression of heat shock protein 70 mRNA and protein after heat treatment in cells of aged rats.

The effect of aging on the induction of heat shock protein 70 (HSP70)-encoding gene expression by elevated temperatures was studied in cultures of lung- or skin-derived fibroblasts from young (5 mo) and old (24 mo) male Wistar rats. Although the kinetics of the heat shock response were found to be similar in the two age groups, we observed lower levels of induction of HSP70 mRNA and HSP70 protein in confluent primary lung and skin fibroblast cultures derived from aged animals. Additional experiments with freshly excised lung tissue showed a similar age-related decline in the heat-induced expression of HSP70.

Aging↗
PubMed1989

Mammalian genes coordinately regulated by growth arrest signals and DNA-damaging agents.

More than 20 different cDNA clones encoding DNA-damage-inducible transcripts in rodent cells have recently been isolated by hybridization subtraction (A. J. Fornace, Jr., I. Alamo, Jr., and M. C. Hollander, Proc. Natl. Acad. Sci. USA 85:8800-8804, 1988). In most cells, one effect of DNA damage is the transient inhibition of DNA synthesis and cell growth. We now show that five of our clones encode transcripts that are increased by other growth cessation signals: growth arrest by serum reduction, medium depletion, contact inhibition, or a 24-h exposure to hydroxyurea. The genes coding for these transcripts have been designated gadd (growth arrest and DNA damage inducible). Two of the gadd cDNA clones were found to hybridize at high stringency to transcripts from human cells that were induced after growth cessation signals or treatment with DNA-damaging agents, which indicates that these responses have been conserved during mammalian evolution. In contrast to results with growth-arrested cells that still had the capacity to grow after removal of the growth arrest conditions, no induction occurred in HL60 cells when growth arrest was produced by terminal differentiation, indicating that only certain kinds of growth cessation signals induce these genes. All of our experiments suggest that the gadd genes are coordinately regulated: the kinetics of induction for all five transcripts were similar; in addition, overexpression of gadd genes was found in homozygous deletion c14CoS/c14CoS mice that are missing a small portion of chromosome 7, suggesting that a trans-acting factor encoded by a gene in this deleted portion is a negative effector of the gadd genes. The gadd genes may represent part of a novel regulatory pathway involved in the negative control of mammalian cell growth.

Amino Acid Sequence↗
PubMed1987

In situ detection of enzymes in yeast.

A very simple method that allows for the rapid in situ assay of enzyme activity in yeast is described. Single colonies are collected on sticks (or glass micropipets), or multiple colonies are collected on sandpaper, and crushed onto nitrocellulose filters. The filters in turn are stained for the enzyme of interest using a histochemical assay. The method is quantitative and was found to work well for four enzymes in yeast.

Acid Phosphatase↗
PubMed1984

Identification and cytogenetic localization of vitelline membrane messenger RNAs in Drosophila.

During stages 9 and 10 of oogenesis in Drosophila the major proteins involved in vitelline membrane (VM) formation are synthesized and secreted by the somatic follicle cells surrounding the oocyte. To identify potential mRNAs involved in VM protein synthesis, newly synthesized poly(A)-containing RNA from egg chambers of different developmental stages was studied. Urea-agarose gel electrophoresis revealed two RNA bands in stage 10 egg chambers in the size range expected for those which encode the smaller VM proteins. These RNA bands, T1 and T2, are specifically enriched in stage 10 follicle cell preparations. In vitro translations in reticulocyte lysates in the absence and presence of microsomal membranes showed both RNA bands code for products that are synthesized in precursor forms which are processed to species that comigrate with VM proteins. T2 directed the synthesis of processed species that comigrated with the 23- to 24-kDa and 17.5-kDa VM proteins (J. Fargnoli and G.L. Waring, 1982, Dev. Biol. 92, 306-314) while the T1 translation product comigrated with the 14-kDa protein. To determine the cytogenetic location of the genes encoding T1 and T2 RNAs, radiolabeled T1 and T2 RNAs were hybridized in situ to salivary gland chromosomes. The results suggest that the structural genes coding for the small vitelline membrane proteins are localized at two sites on the second chromosome: 39DE and 42A.

Animals↗