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.     

Mosquito population structure,pathogen surveillance and insecticide resistance monitoring in urban regions of Crete,Greece

BackgroundIn Greece vector borne diseases (VBD) and foremost West Nile virus (WNV) pose an important threat to public health and the tourist industry, the primary sector of contribution to the national economy. The island of Crete, is one of Greece’s major tourist destinations receiving annually over 5 million tourists making regional VBD control both a public health and economic priority.MethodologyUnder the auspices of the Region of Crete, a systematic integrative surveillance network targeting mosquitoes and associated pathogens was established in Crete for the years 2018–2020. Using conventional and molecular diagnostic tools we investigated the mosquito species composition and population dynamics, pathogen infection occurrences in vector populations and in sentinel chickens, and the insecticide resistance status of the major vector species.Principal findingsImportant disease vectors were recorded across the island including Culex pipiens, Aedes albopictus, and Anopheles superpictus. Over 75% of the sampled specimens were collected in the western prefectures potentially attributed to the local precipitation patterns, with Cx. pipiens being the most dominant species. Although no pathogens (flaviviruses) were detected in the analysed mosquito specimens, chicken blood serum analyses recorded a 1.7% WNV antibody detection rate in the 2018 samples. Notably detection of the first WNV positive chicken preceded human WNV occurrence in the same region by approximately two weeks. The chitin synthase mutation I1043F (associated with high diflubenzuron resistance) was recorded at an 8% allelic frequency in Lasithi prefecture Cx. pipiens mosquitoes (sampled in 2020) for the first time in Greece. Markedly, Cx. pipiens populations in all four prefectures were found harboring the kdr mutations L1014F/C/S (associated with pyrethroid resistance) at a close to fixation rate, with mutation L1014C being the most commonly found allele (≥74% representation). Voltage gated sodium channel analyses in Ae. albopictus revealed the presence of the kdr mutations F1534C and I1532T (associated with putative mild pyrethroid resistance phenotypes) yet absence of V1016G. Allele F1534C was recorded in all prefectures (at an allelic frequency range of 25–46.6%) while I1532T was detected in populations from Chania, Rethymnon and Heraklion (at frequencies below 7.1%). Finally, no kdr mutations were detected in the Anopheles specimens included in the analyses.Conclusions/SignificanceThe findings of our study are of major concern for VBD control in Crete, highlighting (i) the necessity for establishing seasonal integrated entomological/pathogen surveillance programs, supporting the design of targeted vector control responses and; ii) the need for establishing appropriate insecticide resistance management programs ensuring the efficacy and sustainable use of DFB and pyrethroid based products in vector control.     

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.