A Neurotoxic Insecticide Promotes Fungal Infection in Aedes aegypti Larvae by Altering the Bacterial Community.

Symbiotic bacteria have a significant impact on the formation of defensive mechanisms against fungal pathogens and insecticides. The microbiome of the mosquito Aedes aegypti has been well studied; however, there are no data on the influence of insecticides and pathogenic fungi on its structure. The fungus Metarhizium robertsii and a neurotoxic insecticide (avermectin complex) interact synergistically, and the colonization of larvae with hyphal bodies is observed after fungal and combined (conidia + avermectins) treatments. The changes in the bacterial communities (16S rRNA) of Ae. aegypti larvae under the influence of fungal infection, avermectin toxicosis, and their combination were studied. In addition, we studied the interactions between the fungus and the predominant cultivable bacteria in vitro and in vivo after the coinfection of the larvae. Avermectins increased the total bacterial load and diversity. The fungus decreased the diversity and insignificantly increased the bacterial load. Importantly, avermectins reduced the relative abundance of Microbacterium (Actinobacteria), which exhibited a strong antagonistic effect towards the fungus in in vitro and in vivo assays. The avermectin treatment led to an increased abundance of Chryseobacterium (Flavobacteria), which exerted a neutral effect on mycosis development. In addition, avermectin treatment led to an elevation of some subdominant bacteria (Pseudomonas) that interacted synergistically with the fungus. We suggest that avermectins change the bacterial community to favor the development of fungal infection.

Identification of new M23A mRNA of mouse aquaporin-4 expressed in brain, liver, and kidney.

Aquaporins (AQPs) belong to a transmembrane protein family of water channels that are permeable to water by the osmotic gradient. There are two isoforms of mouse AQP4 - M1 and M23. Their balance in the cell determines water permeability of the plasma membrane. These two isoforms are encoded by three mRNAs: M1 isoform is encoded by M1 mRNA and M23 isoform is encoded by M23 and M23X mRNAs. Here we found a new fourth mRNA of mouse AQP4 - M23A mRNA. The start of transcription is different for M23A mRNA from all the known AQP4 mRNAs. The 5'-untranslated region (5'-UTR) of M23A mRNA is encoded by four new exons (A, B, C, and D), which are located in the 5' region from exon-0 of the AQP4 gene. Alternative splicing between the exons-A, -B, -C, and -D leads to formation of multiple variants of M23A mRNA. We cloned six of these variants, all of which code full length M23 isoform of AQP4. Using RT-PCR we detected tissue-specific expression of the new M23A and already known M23, M23X, and M1 mRNAs. The M23A mRNA is expressed mostly in kidney, liver, and brain. Analysis of mRNA 5'-UTR structure showed low translation efficacy for M1 mRNA in comparison with high translation efficacy for M23A, M23X, and M23 mRNAs. We propose that AQP4 expression is controlled tissue-specifically by independent promoters. Thus multiple AQP4 mRNAs may allow long-term regulation of the balance between M1 and M23 AQP4 isoforms in the cell and thus water permeability of the plasma membrane.

Identification of a new form of AQP4 mRNA that is developmentally expressed in mouse brain.

The water channel aquaporin 4 (AQP4) is abundantly expressed in the brain, and also in lung and kidney. Previous studies have suggested that there are at least two AQP4 mRNA. The two mRNA encode for two AQP4 proteins that differ with regard to the length of the N-terminal: AQP4.M1 and AQP4.M23. Here we report, by use of reverse transcriptase PCR and comparison of genomic and cDNA structures, the presence of a third form of mouse AQP4 mRNA. The upstream sequence of this form of mRNA originates from an additional exon, interspaced between exon 0 and exon 1, and an alternatively spliced form of exon 1. Analysis of nucleotide sequence suggests that this new form of AQP4 mRNA also encodes for the AQP4.M23 protein. The two forms of AQP4 mRNA that presumably both encode for M23 have a tissue- and age-specific expression. The new AQP4 mRNA was predominantly expressed in brain. The expression was approximately twofold higher in the adult brain than in the infant brain. In contrast, the expression levels of the new mRNA were low in both infant and adult lung and kidney. The previously described mRNA encoding for AQP4.M23 was predominantly expressed in lung and kidney. In lung, the expression of this form was higher in infancy than in adulthood. In conclusion, we have identified a new form of AQP4 mRNA that is predominantly expressed in the brain and that is developmentally regulated.

[Highly selective labeling of the E. coli RNA-polymerase promoter complex with reactive derivatives of oligonucleotide primers of various specificity]. / Vysokoselektivnoe mechenie kompleksa RNK-polimerazy E. coli s promotorom reaktsionnosposobnymi proizvodnymi oligonukleotidov- zatravok razlichnoi spetsifichnosti.

A technique of highly selective affinity labelling, which includes covalent modification of the enzyme-T7A2 promoter complex with reactive oligonucleotide derivatives and subsequent elongation of the attached oligonucleotide residue with a radioactive substrate was used to study the product-binding site of E. coli RNA polymerase. Different oligonucleotides complementary to the T7A2 promoter (with lengths ranging from 2 to 8 residues) containing 5'-terminal phosphorylating, alkylating or aldehyde groups were used for the labelling. The procedure resulted in labelling DNA and beta-, beta'- or sigma-subunits of the enzyme, which are therefore believed to contact with growing RNA in the course of initiation. Consideration of the labelling patterns as a functions of the oligonucleotide's length as well as of the structure and chemical specificity of the reactive groups led to a tentative topographic scheme of the RNA polymerase product-binding region.