Inhibitory action of different RNA fractions on the translation of globin mRNA in vitro.

Preparations of 28S rRNA and 12S RNA, obtained from sheep lymphocytes, were shown to inhibit the translation of globin mRNA. An inhibition by a given amount of 12S or 28S RNA was only obvious at saturating or near saturating levels of globin mRNA, suggesting that the inhibitory RNAs interacted with some factor essential for protein synthesis other than mRNA. The inhibitory RNAs had no effect on the translation of the synthetic template polyuridylic acid. It is suggested therefore that the target for inhibitory RNAs might be a natural mRNA specific initiation factor.     

Recombinant Cholinergic Differentiation Factor (Leukemia Inhibitory Factor) Regulates Sympathetic Neuron Phenotype by Alterations in the Size and Amounts of Neuropeptide mRNAs

The cholinergic differentiation factor (CDF) in heart cells is identical to leukemia inhibitory factor (LIF). Recombinant CDF/LIF was shown to alter dramatically neurotransmitter production as well as the levels of several neuropeptides in cultured rat sympathetic neurons. Here it is shown that these changes are likely to be caused by alterations in the mRNA for these proteins and peptides. Growth in 1 nM recombinant CDF/LIF induces mRNA for acetyl CoA: choline-O-acetyltransferase [EC 2.3.1.6; choline acetyltransferase (ChAT)], somatostatin (SOM), substance P, and vasoactive intestinal polypeptide while lowering mRNA levels of tyrosine hydroxylase (EC 1.14.16.2) and neuropeptide Y (NPY). In addition, the sizes of the mRNAs for ChAT, SOM, and NPY are larger after recombinant CDF/LIF treatment.     

Expression of rat transforming growth factor alpha mRNA during development occurs predominantly in the maternal decidua.

Previous studies have shown that transforming growth factor alpha is expressed during rodent development. To establish the site(s) of transforming growth factor alpha mRNA expression during rat embryogensis, we performed in situ hybridization and Northern blot analyses on samples of embryonic and maternal tissues at various gestational ages. Our results indicate that the high levels of transforming growth factor alpha mRNA that are observed during early development are the result of expression in the maternal decidua and not in the embryo. Decidual expression appears to be induced after implantation, peaks at day 8, and then slowly declines through day 15 at which time the decidua is being resorbed. Expression of transforming growth factor alpha mRNA is highest in that region of the decidua adjacent to the embryo and is low or nondetectable in the uterus, placenta, and other maternal tissues. The developmentally regulated expression of transforming growth factor alpha mRNA in the decidua, together with the presence of epidermal growth factor receptors in this tissue, suggests that transforming growth factor alpha stimulates proliferation locally through an autocrine mechanism. Since epidermal growth factor receptors are present in the embryo and placenta, transforming growth factor alpha produced in the decidua may also act on these tissues through paracrine or endocrine mechanisms.     

RT-PCR method to quantify vascular endothelial growth factor expression.

The vascular endothelial growth factor (VEGF) is implicated in the progression of cancers. Its expression is well correlated with tumor growth and metastases. The availability of a rapid and sensitive method to detect the amounts of VEGF mRNA in biological samples of limited size, very small biopsies, or samples containing relatively few cells could provide an interesting prognostic tool for clinicians. We have developed an RT-PCR method that allows us to detect the VEGF mRNA from as little as 3 micrograms total mRNA. We have also shown that this protocol can be generalized to all cell lines tested. This method constitutes a very potent tool for the analysis of VEGF mRNA expression in different contexts.     

The viral protein sigma 3 participates in translation of late viral mRNA in reovirus-infected L cells.

Reovirus late (uncapped) mRNA was previously shown to be efficiently translated in vitro extracts prepared from infected cells but not from uninfected cells. We demonstrated that different fractions from infected cells can stimulate translation of late viral mRNA when added to uninfected extracts. The activity of the different fractions correlated with their relative content of the sigma 3 capsid protein; the fraction prepared by high-salt wash of the ribosomes had the highest specific activity. The activity present in this fraction was abolished by preincubation with an anti-sigma 3 serum. Purified sigma 3 protein also stimulated the translation of late viral mRNA, confirming that it was the factor involved. Altogether, these results suggest that this protein plays the role of a late-viral-mRNA-specific initiation factor. The absence of an inhibitory effect of sigma 3 on the translation of other mRNAs indicates that this protein is not directly involved in the inhibition of host and early viral mRNA translation that occurs in infected cells but that a second mechanism is probably operative.     

Down-regulated expression of TWEAK mRNA in acute and chronic inflammatory pathologies

TWEAK is a newly identified member of the Tumor Necrosis Factor (TNF) family of proteins which are involved in many immunoinflammatory mechanisms. The putative role of TWEAK in inflammation was analyzed in mice treated with lipopolysaccharide (LPS), a strong inducer of the immuno-inflammatory responses. TWEAK mRNA rapidly disappeared in all the tissues tested. Analysis of LPS-treated thioglycolate-elicited peritoneal macrophages revealed that the rapid loss of TWEAK mRNA was due to its active destabilization. In chronic pathologies like autoimmune hemolytic anemia in the NZB mouse strain or systemic lupus erythematosus (SLE) in the BXSB mouse strain, TWEAK mRNA was shown to be reduced concomitantly to the development of chronic autoimmune diseases. These results demonstrated that TWEAK mRNA, contrary to TNF mRNA, is stable, ubiquitously distributed in tissues, and is down-regulated after LPS treatment or in chronic inflammation, suggesting that TWEAK could be an important factor, along with TNF, in acute and chronic inflammations.     

Marked induction of sterol 27-hydroxylase activity and mRNA levels during differentiation of human cultured monocytes into macrophages

Sterol 27-hydroxylase has been suggested to be involved in an alternative pathway for the elimination of cholesterol from macrophages and early atherosclerotic lesions. We have previously shown that human lung macrophages as well as monocyte-derived macrophages have a relatively high activity of sterol 27-hydroxylase (CYP27). This enzyme converts intracellular cholesterol into 27-hydroxycholesterol and cholestenoic acid that flux from cultured cells into the medium. It is shown here that human monocytes have very low CYP27 activity and CYP27 mRNA levels. During differentiation into macrophages, both CYP27 activity and CYP27 mRNA levels increase markedly after 4 days of culture in serum-free medium. Addition of macrophage-colony stimulating factor had no significant effect on the induction and addition of fetal calf serum had an inhibitory effect. Cholesterol synthesis was found to be a critical factor for the production of 27-oxygenated products by the macrophages cultured in serum-free medium. The increased capacity of the differentiated cells to eliminate intracellular cholesterol is of interest and supports the contention that CYP27 is an antiatherogenic factor.     

MAPKK-dependent growth factor-induced upregulation of P2Y2 receptors in vascular smooth muscle cells.

The ATP- and UTP-sensitive P2Y2 receptor which mediates both contractile and mitogenic effects has recently been shown to be upregulated in the synthetic phenotype of the vascular smooth muscle cell (VSMC). Using a competitive RT-PCR we demonstrate that the P2Y2 receptor mRNA is increased by fetal calf serum and other growth factors in a MAPKK-dependent way. This was confirmed at the functional level by examining UTP-stimulated release of intracellular Ca2+. Furthermore, the P2Y2 receptor mRNA is positively autoregulated by ATP and the mRNA is rapidly degraded with only 26% remaining after 1 h in the presence of actinomycin D. Our results indicate growth factor regulation and rapid turnover of the P2Y2 receptor mRNA, which may be of importance in atherosclerosis and neointima formation after balloon angioplasty.     

The short form of heparin-binding EGF-like growth factor. Part II

The structure of monkey (Chlorocebus aethiops) heparin-binding EGF-like growth factor (HB-EGF) gene has been investigated in this work in comparison with the known structure of human gene. It was shown that HB-EGF short form (SF-HB-EGF) specific exon 3a is mapped between exons 3 and 4 at distance 700 b.p. from exon 4. In a number of human and simian cell lines the main part of SF-HB-EGF mRNA does not contain HB-EGF mRNA specific exons 4 and 5. In comparison with HB-EGF mRNA in SF-HB-EGF mRNA P-form, but not L-form of is predominant, and this mRNA encodes a polypeptide with changed propeptide structure. Labeled SF-HB-EGF competes with HB-EGF and EGF for binding sites at A431 cell surface, which may be due to interaction with specific receptor. All the data suggest a specific role of SF-HB-EGF in cellular signalization.     

The causes of protein evolutionary rate variation

The rate of protein evolution varies more than 1000-fold and, for the past 30 years, it was thought that the rate was determined by protein function. Drummond and co-workers have now shown that a single factor underlying mRNA expression, protein abundance and synonymous codon usage is the chief causal agent of protein evolutionary rate in yeast. It will be interesting to see whether this is shown to be a universal rule for all biological systems.     

Human adipsin is identical to complement factor D and is expressed at high levels in adipose tissue.

A cDNA for human adipsin was isolated and shown to encode a protein sharing 98% amino acid sequence similarity with the protein sequence previously determined for purified natural human complement factor D. Like mouse adipsin, recombinant human adipsin displays the enzymatic activity of human complement factor D, cleaving complement factor B only when B is complexed with activated complement component C3. We conclude that human adipsin is equivalent to complement factor D and that adipsin is the homologue of factor D in rodents. Adipose tissue is a major site of synthesis of human adipsin/complement factor D mRNA, but unlike the case in rodents, human adipsin mRNA is also expressed in monocytes/macrophages. The data presented here, demonstrating the equivalence of human adipsin to complement factor D and its high level of expression in fat, suggest a previously unsuspected role for adipose tissue in immune system biology.     

Regulation of thymidylate synthase gene expression in mouse fibroblasts synchronized by mitotic selection

Previous studies have shown that thymidylate synthase gene expression is regulated over a wide range in response to growth stimulation in cultured mouse fibroblasts. In the present study we show that the gene is also regulated during the cell cycle in continuously growing cells. Our analyses were conducted with a fluorodeoxyuridine-resistant mouse 3T6 cell line that overproduces thymidylate synthase and its mRNA by a factor of 50 due to gene amplification. Cells were synchronized by mitotic selection. RNA blot analyses showed that the amount of thymidylate synthase mRNA increased 5- to 10-fold as cells progressed from G1 through the middle of S phase. S1 nuclease protection assays showed that the pattern of 5' termini of thymidylate synthase mRNA was the same in G1 and S phase. Despite the large increase in thymidylate synthase mRNA content, the level of the enzyme increased only by a factor of 2 as cells progressed from G1 to mid S phase. This apparent discrepancy can be explained by the fact that the enzyme is highly stable.