Induction of cytogenetic damage by modified steroidal derivatives of p-bis(2-chloroethyl)aminophenylacetic acid in human lymphocytes

The effect of modified steroids, containing alkylating agents, on SCE rates and on cell kinetics in cultured human lymphocytes was studied. The homo-aza-steroidal ester of p-bis(2-chloroethyl)aminophenylacetic acid (ASE) was found to be the most effective in causing markedly increased SCE rates and cell division delays. The androsterone ester of p-bis(2-chloroethyl)aminophenylacetic acid (AE-CAPA) was found to be next in order of effectiveness with the lactone ester (LE-CAPA), chlorambucil ester 3 beta-hydroxy-13a-amino-13,17-seco-5a-androstan-17-oic-13,17-lactam (CBC-HAAL) and chlorambucil (CBC) following. p-Bis(2-chloroethyl)aminophenylacetic acid (CAPA) had only a small effect and 3 beta-hydroxy-13a-amino-13,17-seco-5a-androstan-17-oic-13,17-lactam (HAAL) had no effect at all. A correlation between potency for SCE induction, effectiveness in cell division delay and previously established antitumor activity of these drugs was observed.     

Enhancement of cytogenetic damage by chlorpromazine in human lymphocytes treated with alkylating antineoplastics and caffeine

In cultured human lymphocytes chlorpromazine (CPZ) was found to induce cell division delays and to have no effect on sister-chromatid exchanges (SCEs) or on mitotic indices (MIs). CPZ induces cytotoxic effects in combination with caffeine (CAF) and alkylating agents. In combination with CAF it induced cell division delays and suppression of MIs. In combination with melphalan (MEL) and CAF, CPZ synergistically induced SCEs, caused cell division delay and suppressed MIs. In combination with chlorambucil (CBC) and CAF, CPZ produced synergism on induction of SCEs, enhanced cell division delays and reduced MIs.     

miRNP:mRNA association in polyribosomes in a human neuronal cell line

MicroRNAs (miRNAs) are small regulatory RNAs that control gene expression by base-pairing with their mRNA targets. miRNAs assemble into ribonucleoprotein complexes termed miRNPs. Animal miRNAs recognize their mRNA targets via partial antisense complementarity and repress mRNA translation at a step after translation initiation. How animal miRNAs recognize their mRNA targets and how they control their translation is unknown. Here we describe that in a human neuronal cell line, the miRNP proteins eIF2C2 (a member of the Argonaute family of proteins), Gemin3, and Gemin4 along with miRNAs cosediment with polyribosomes. Furthermore, we describe a physical association between a let-7b (miRNA)-containing miRNP and its putative human mRNA target in polyribosome-containing fractions. These findings suggest that miRNP proteins may play important roles in target mRNA recognition and translational repression.     

A new approach for evaluating in vivo anti-leukemic activity using the SCE assay. An application on three newly synthesised anti-tumour steroidal esters

Three newly synthesised steroidal esteric derivatives of nitrogen mustard (compounds 1-3) were comparatively studied on a molar basis regarding their ability to induce sister chromatid exchanges (SCEs) in normal human lymphocytes in vitro and therapeutic effects on leukemia P388 bearing mice. Compounds 1 and 3 are modified steroidal esters of p-methyl-m-N,N-bis(2-chloroethyl)amino benzoic acid, and compound 2 is a modified steroidal ester of chlorambucil. All compounds induced statistically significant increases in SCEs and decreases in proliferation rate indices (PRIs) of cultured human lymphocytes and significantly increased the life span of P388 bearing mice. In this study, the doses applied for therapeutic purposes upon leukemia P388 bearing mice in vivo were derived from cytogenetic observations in normal human lymphocytes in vitro. A substantially better therapeutic effect was obtained compared to the effect achieved after the use of quite higher doses related with LD(10) values. We have demonstrated that the order of anti-tumour effectiveness of the treatment schedules of the three newly synthesised compounds tested (at doses derived from cytogenetic observations) coincides with the order of the cytogenetic effects they induce. The SCE assay appears to have an application in the clinical prediction of tumour sensitivity to potential chemotherapeutics.     

Numerous microRNPs in neuronal cells containing novel microRNAs

Spinal muscular atrophy (SMA) is a common neurodegenerative disease that is caused by deletions or loss-of-function mutations in the Survival of Motor Neuron (SMN) protein. SMN is part of a large complex that functions in the assembly/restructuring of ribonucleoprotein (RNP) complexes. We recently showed in HeLa cells that two components of the SMN complex, Gemin3 and Gemin4, together with the argonaute protein eIF2C2, also associate with microRNAs (miRNAs) as part of a novel class of RNPs termed miRNPs. Here we report on miRNPs isolated from neuronal cell lines of mouse and human, and describe 53 novel miRNAs. Several of these miRNAs are conserved in divergent organisms, including rat, zebrafish, pufferfish, and the nematode Caenorhabditis elegans. The chromosomal locations of most of the novel miRNAs were identified and indicate some phylogenetic conservation of the likely precursor structures. Interestingly the gene locus of one miRNA, miR-175, is a candidate region for two neurologic diseases: early-onset parkinsonism (Waisman syndrome) and X-linked mental retardation (MRX3). Also, several miRNAs identified as part of miRNPs in these cells appear to constitute two distinct subfamilies. These subfamilies comprise multiple copies of miRNAs on different chromosomes, suggesting an important function in the regulation of gene expression.     

RAKE and LNA-ISH reveal microRNA expression and localization in archival human brain

microRNAs (miRNAs) are small (approximately 22 nucleotide) regulatory RNAs which play fundamental roles in many biological processes. Recent studies have shown that the expression of many miRNAs is altered in various human tumors and some miRNAs may function as oncogenes or tumor suppressor genes. However, with the exception of glioblastoma multiforme, the expression of miRNAs in brain tumors is unknown. Furthermore, methods to profile miRNAs from formalin-fixed, paraffin-embedded (FFPE) archival tissues or to study their cellular and subcellular localization in FFPE tissues have been lacking. Here we report the coordinated miRNA expression analysis from the tissue level to the subcellular level, using the RAKE (RNA-primed, array-based, Klenow Enzyme) miRNA microarray platform in conjunction with Locked Nucleic Acid (LNA)-based in situ hybridization (LNA-ISH) on archival FFPE human brains and oligodendroglial tumors. The ability to profile miRNAs from archival tissues at the tissue level, by RAKE microarrays, and at the cellular level by LNA-ISH, will accelerate studies of miRNAs in human diseases.     

Immunochemical characterization of polysaccharide antigens from six clinical strains of Enterococci

Background  

Enterococci have become major nosocomial pathogens due to their intrinsic and acquired resistance to a broad spectrum of antibiotics. Their increasing drug resistance prompts us to search for prominent antigens to develop vaccines against enterococci. Given the success of polysaccharide-based vaccines against various bacterial pathogens, we isolated and characterized the immunochemical properties of polysaccharide antigens from five strains of Enterococcus faecalis and one strain of vancomycin-resistant E. faecium.     

A new technique for long preservation of 14C-labelled Cladocerans

Three ways of preserving labelled Cladocerans fed with ¹⁴C-Chlorella for 7.5–10 min were tested. Tracer leakage in 4% formalin at room temperature is rapid and extensive (half of the label was found in the animals after 1 hour of preservation). Even when individuals are frozen and sorting is made quickly in a liquid, losses nevertheless occur (substantial decrease of animal activity after only 4–5 min in the water in one of the two experiments performed). Results obtained after freezing in 4% formalin and sorting exactly 2 hours after thawing gave consistent losses: 16 separate experiments with Daphnia pulex, Ceriodaphnia spp. and Diaphanosoma brachyurum gave apparent filtering rates underestimated from 35% to 63% for freezing periods of up to 45 days. The good agreement in in situ community filtering rates between measured values and estimated ones from individual data confirmed the validity of a correction factor of x 2 applied to animals frozen in formalin.     

Enhancement by methylxanthines of sister-chromatid exchange frequency induced by cytostatics in normal and leukemic human lymphocytes

The effect of theobromine (TB) and diphylline (DP) or (1,2-dihydroxy-3-propyl)theophylline on SCE rates induced in vitro by mitomycin C (MMC), and the effect of caffeine on SCE rates induced in vitro by cytosine arabinoside (Ara-C) was studied. The combined treatments with MMC plus TB or DP showed the potentiating ability of the latter drugs. Caffeine also enhanced SCEs induced by Ara-C in cultured human lymphocytes. Caffeine and adriamycin (ADR) did not act synergistically on induction of SCEs. In a combined study, in vivo and in vitro, lymphocytes taken from 2 leukemic patients who had been given chlorambucil (CBC) or Ara-C by injection 3 h before, and then treated with caffeine in vitro, were found to have synergistically increased exchange frequencies.