Regulating a translational regulator: mechanisms cells use to control the activity of the fragile X mental retardation protein.

Fragile X syndrome results from the loss of a normal cellular protein, FMRP. FMRP is an RNA binding protein, and it is likely that altering the way FMRP's messenger RNA (mRNA) targets are processed results in the clinical features associated with the disease. Using complementary DNA microarray screening, a number of brain-derived mRNAs that interact directly with FMRP in vitro and associate with FMRP-containing mRNPs in vivo have been identified. These target messages encode RNA-binding proteins, transcription factors, neuronal receptors, cytoskeletal proteins, a few enzymes as well as several unknown proteins. For a subset of these mRNAs it has been shown that modulating FMRP levels in cultured cells correspondingly affects their expression. In addition, several modes by which cells modulate FMRP activity have been described; these include posttranscriptional processing and posttranslational modification. Here, the most recent results concerning the biochemical activities of FMRP and how they are affected by various modifications are reviewed. The data lead to a model signaling mechanism by which FMRP normally regulates the expression of its target mRNAs.

Significance of sexual route of transmission of hepatitis B and C in Russia.

It is generally understood that hepatitis B and hepatitis C may be sexually transmitted. During the last decade there was a sharp growth of hepatitis B and C in Russia. In comparison to 1992 the incidence of hepatitis B in Russia rose two-fold and in 1999 there were 43.31 cases per 100,000 of population (in some cities up to 150/100,000 and even more). The incidence of hepatitis C in 1999 (19.31 per 100,000 of population) rose to six times more than in 1994. At the same time there was a dramatic growth in syphilis and other sexually transmitted infections in Russia. The proportion of sexual transmission of hepatitis B virus (HBV) and hepatitis C virus (HCV) compared with other routes of transmission increased. According to the data from Moscow City Centre of Epidemiology during the last two years, up to 40% cases of HCV and HBV were sexually transmitted. The most dramatic growth of registered cases of hepatitis was seen among the sexually active population aged 14-29. Confirmation of the sexual route of transmission of HBV and HCV in teenagers was seen when the results of the study showed various markers of HBV and HCV to be significantly more common among sexually active (n = 45) than sexually inactive (n = 341) teenagers (13.33% vs. 4.39% for HBsAg; 46.67% vs. 12.61% for HBsAg+anti-HBs+anti-HBc; 9.47% vs. 3.95% for anti-HCV, respectively).

Self-cleaving-ribozyme-mediated reduction of betaAPP in human rhabdomyosarcoma cells.

A self-cleaving hammerhead ribozyme targeted to codon 47 in beta-amyloid precursor protein (betaAPP) mRNA was cloned as a eucaryotic transcription cassette into the 3' UTR of enhanced green fluorescence protein (EGFP) mRNA, producing a C-terminal fusion mRNA. CMV promotor-driven vectors bearing this construct or a mutationally inactive ribozyme construct were transiently transfected into human embryonic rhabdomyosarcoma (A-204) cells and their effects studied. Ribozyme self-cleavage in vivo was demonstrated by Northern blotting and the site of self-cleavage was delineated using site-specific deoxyoligonucleotide probes and primer extension arrest. Using this ribozyme reporter we demonstrated that ribozyme expression correlated with lower betaAPP levels in the transfected cells. Control studies with the inactive ribozyme construct showed that both ribozyme cleavage and antisense mechanisms combined to produce the observed effect. Furthermore, production of truncated EGFP mRNA via ribozyme self-cleavage reduced EGFP-reporter expression compared to full-length EGFP control mRNAs, indicating that truncation affects the translatability of the reporter. This occurred because of a slight decrease in the stability of the fusion mRNA. The results of these studies suggest that self-cleaving ribozyme vectors may be an effective means of delivering and visualizing the expression of small active ribozymes in vivo.

In vivo ribozyme targeting of betaAPP+ mRNAs.

In Alzheimer's disease (AD) and Down's syndrome (DS) patients, posttranscriptional alterations of sequences encoded by exon 9 and exon 10 of the beta-amyloid precursor protein (betaAPP) mRNA result in mutant proteins (betaAPP+) that colocalize with neurofibrillary tangles and senile plaques. These aberrant messages may contribute to the development of sporadic or late-onset Alzheimer's disease; thus, eliminating them or attenuating their expression could significantly benefit AD patients. In the present work, self-cleaving hammerhead ribozymes targeted to betaAPP exon 9 (Rz9) and betaAPP+ mutant exon 10 (Rz10) were examined for their ability to distinguish between betaAPP and betaAPP+ mRNA. In transiently transfected A-204 cells, quantitative confocal fluorescence microscopy showed that Rz9 preferentially lowered endogenous betaAPP. In contrast, in transient cotransfection experiments with betaAPP+ mRNAs containing a wild-type exon 9 and mutant exon 10 (betaAPP-9/betaAPP-10+1), or a mutant exon 9 and wild-type exon 10 (betaAPP-9+1/betaAPP-10) we found that Rz9 and Rz10 preferentially reduced betaAPP+ -mutant exon 10 mRNA in a concentration and a ribozyme-dependent manner.

Isolation and properties of lead-resistant variants of rat glioma cells.

Glial cells are thought to protect neurons from heavy-metal toxicity. To gain a better understanding of mechanisms of protection against lead compounds, a number of lead-resistant C6 rat glioma cell sublines have been isolated. After 8 mo of growth in the absence of lead nitrate, three sublines still maintain their lead-resistant phenotype. None of the lead-resistant sublines are cross-resistant to Cd(II) or Ni(II), but all are cross-resistant (in varying degrees) to Hg(II), As(III), Sb(III), and Sn(II), and one is resistant to trimethyl tin. No inducible lead resistance is seen in any glioma line. One subline has been used to create cell-cell hybrids with wild-type cells. The hybrids exhibit dominance of the lead-resistant phenotype. To identify and analyze altered gene expression at the mRNA level in the lead-resistant sublines, the differential display technique was used. Numerous differences are seen between amplified fragments from wild-type and lead-resistant cells. Candidate clones are now being analyzed to confirm the differential expression and to isolate cDNAs that confer lead resistance.

Chinese hamster cells expressing antisense to metallothionein become spontaneous mutators.

The functions of metallothioneins (MTs) have been debated for at least a decade. Because it seems unlikely that they evolved only to protect cells against exogenous heavy metals, it has been suggested that MTs have roles in scavenging reactive intermediates, controlling zinc and copper homeostasis, and controlling transfer of zinc to transcription factors and other proteins. Previously, we demonstrated that Chinese hamster G12 cells which overexpress MT have greatly reduced spontaneous mutation rates, suggesting that MT evolved to prevent spontaneous mutagenesis induced by free nuclear zinc ions. We have now isolated G12 transfectants which express antisense RNA to MT. Immunofluorescent staining reveals MT protein in both the nucleus and the cytoplasm in parental cells. A clone expressing high levels of antisense RNA (AMT30) shows reduced basal and induced levels of MT protein. AMT30 cells are hypersensitive to cadmium, zinc, copper and mercury chlorides as well as to menadione. Glutathione levels in AMT30 and G12 cells do not differ. AMT30 cells are spontaneous mutators, showing a spontaneous mutation rate 5-10 times that of G12 cells or G12 cells transfected with vector alone. Only transfectants which show a high level of MT antisense expression (i.e., AMT30) had greatly elevated spontaneous mutation rates. These results support our hypothesis that a major role of MT is to act as an endogenous antimutagen probably via scavenging of reactive intermediates in the nucleus. AMT30 cells should be useful in delineating the sources of spontaneous mutagenesis.