A yeast-endonuclease-generated DNA break induces antigenic switching in Trypanosoma brucei.

Trypanosoma brucei is the causative agent of African sleeping sickness in humans and one of the causes of nagana in cattle. This protozoan parasite evades the host immune system by antigenic variation, a periodic switching of its variant surface glycoprotein (VSG) coat. VSG switching is spontaneous and occurs at a rate of about 10(-2)-10(-3) per population doubling in recent isolates from nature, but at a markedly reduced rate (10(-5)-10(-6)) in laboratory-adapted strains. VSG switching is thought to occur predominantly through gene conversion, a form of homologous recombination initiated by a DNA lesion that is used by other pathogens (for example, Candida albicans, Borrelia sp. and Neisseria gonorrhoeae) to generate surface protein diversity, and by B lymphocytes of the vertebrate immune system to generate antibody diversity. Very little is known about the molecular mechanism of VSG switching in T. brucei. Here we demonstrate that the introduction of a DNA double-stranded break (DSB) adjacent to the approximately 70-base-pair (bp) repeats upstream of the transcribed VSG gene increases switching in vitro approximately 250-fold, producing switched clones with a frequency and features similar to those generated early in an infection. We were also able to detect spontaneous DSBs within the 70-bp repeats upstream of the actively transcribed VSG gene, indicating that a DSB is a natural intermediate of VSG gene conversion and that VSG switching is the result of the resolution of this DSB by break-induced replication.

A new concept of biocatalytic synthesis of acrylic monomers for obtaining water-soluble acrylic heteropolymers.

Rhodococcus strains were designed as model biocatalysts (BCs) for the production of acrylic acid and mixtures of acrylic monomers consisting of acrylamide, acrylic acid, and N-alkylacrylamide (N-isopropylacrylamide). To obtain BC strains, we used, among other approaches, adaptive laboratory evolution (ALE), based on the use of the metabolic pathway of amide utilization. Whole genome sequencing of the strains obtained after ALE, as well as subsequent targeted gene disruption, identified candidate genes for three new amidases that are promising for the development of BCs for the production of acrylic acid from acrylamide. New BCs had two types of amidase activities, acrylamide-hydrolyzing and acrylamide-transferring, and by varying the ratio of these activities in BCs, it is possible to influence the ratio of monomers in the resulting mixtures. Based on these strains, a prototype of a new technological concept for the biocatalytic synthesis of acrylic monomers was developed for the production of water-soluble acrylic heteropolymers containing valuable N-alkylacrylamide units. In addition to the possibility of obtaining mixtures of different compositions, the advantages of the concept are a single starting reagent (acrylamide), more unification of processes (all processes are based on the same type of biocatalyst), and potentially greater safety for personnel and the environment compared to existing chemical technologies.

Viral induction of AID is independent of the interferon and the Toll-like receptor signaling pathways but requires NF-kappaB.

Activation-induced cytidine deaminase (AID) is expressed in germinal centers of lymphoid organs during immunoglobulin diversification, in bone marrow B cells after infection with Abelson murine leukemia retrovirus (Ab-MLV), and in human B cells after infection by hepatitis C virus. To understand how viruses signal AID induction in the host we asked whether the AID response was abrogated in cells deficient in the interferon pathway or in signaling via the Toll-like receptors. Here we show that AID is not an interferon responsive gene and abrogation of Toll-like receptor signaling does not diminish the AID response. However, we found that NF-kappaB was required for expression of virally induced AID. Since NF-kappaB binds and activates the AID promoter, these results mechanistically link viral infection with AID transcription. Thus, induction of AID by viruses could be the result of several signaling pathways that culminate in NF-kappaB activation, underscoring the versatility of this host defense program.

Orphan receptor NR4A3 is a novel target of p53 that contributes to apoptosis.

Major tumor suppressor and transcription factor p53 coordinates expression of many genes hence affecting critical cellular functions including cell cycle, senescence, and apoptosis. The NR4A family of orphan receptors (NR4A1-3) belongs to the superfamily of nuclear receptors. They regulate genes involved in proliferation, cell migration, and apoptosis. In this study, we report an identification of NR4A3 as a direct transcriptional target of p53. Using various techniques, we showed that p53 directly bound the promoter of NR4A3 gene and induced its transcription. Functionally, over-expression of NR4A3 attenuated proliferation of cancer cells and promoted apoptosis by augmenting the expression of pro-apoptotic genes, PUMA and Bax. Knockdown of NR4A3 reversed these phenotypes. Importantly, NR4A3 exhibited tumor suppressive functions both in p53-dependent and independent manner. In addition, NR4A3 physically interacted with an anti-apoptotic Bcl-2 protein hence sequestering it from blunting apoptosis. These observations were corroborated by the bioinformatics analysis, which demonstrated a correlation between high levels of NR4A3 expression and better survival of breast and lung cancer patients. Collectively, our studies revealed a novel transcriptional target of p53, NR4A3, which triggers apoptosis and thus likely has a tumor suppressive role in breast and lung cancers.

An example of problems in dose reconstruction from doses formed by electromagnetic irradiation by different energy sources.

Dose reconstruction for citizens of Belarus affected by the Chernobyl accident showed an unexpectedly wide range of doses. Using the EPR tooth enamel dosimetry method, it has been demonstrated that when the tooth enamel dose was formed due to x-rays with effective energy of 34 keV and the additional irradiation of enamel samples was performed by gamma radiation with mean energy of 1,250 keV, it led to a considerable increase in the reconstructed absorbed dose as compared with the applied. In the case when the dose was formed due to gamma radiation and the additional irradiation was performed by x-rays, it led to a considerable decrease in the reconstructed dose as compared with the applied. When the dose formation and the additional irradiation were carried out from external sources of electromagnetic radiation of equal energy, the reconstructed dose value was close to that of the applied. The obtained data show that for adequate reconstruction of individual absorbed doses by the EPR tooth enamel spectra, it is necessary to take into account the contribution from diagnostic x-ray examination of the teeth, jaw, and skull of some individuals who were exposed to a combined effect of the external gamma radiation and x-rays.

A role for activation-induced cytidine deaminase in the host response against a transforming retrovirus.

Activation-induced cytidine deaminase (AID) is specifically expressed in the germinal centers of lymphoid organs, where it initiates targeted hypermutation of variable regions of immunoglobulin genes in response to stimulation by antigen. Ectopic expression of AID, however, mediates generalized hypermutation in eukaryotes and prokaryotes. Here, we present evidence that AID is induced outside the germinal center in response to infection by the Abelson murine leukemia virus. The genotoxic activity of virally induced AID resulted in checkpoint kinase-1 (chk1) phosphorylation and ultimately restricted the proliferation of the infected cell. At the same time, it induced NKG2D ligand upregulation, which alerts the immune system to the presence of virally transformed cells. Hence, in addition to its known function in immunoglobulin diversification, AID is active in innate defense against a transforming retrovirus.

Conditional expression of human acid beta-glucosidase improves the visceral phenotype in a Gaucher disease mouse model.

The reversibility and regression of histological and biochemical findings in a mouse model of Gaucher disease (4L/PS-NA) was evaluated using a liver-enriched activator protein promoter control of a tetracycline-controlled transcriptional activation-responsive human acid beta-glucosidase (hGCase) transgenic system. 4L/PS-NA has the acid beta-glucosidase (GCase) V394L/V394L (4L) point mutation combined with hypomorphic ( approximately 6% wild-type) expression of the mouse prosaposin transgene (PS-NA). The hGCase/4L/PS-NA had exclusive liver expression of hGCase controlled by doxycycline (DOX). In the absence of DOX, hGCase was secreted from liver at levels of approximately 120 microg/ml serum with only approximately 8% of full activity, following exposure to pH 7.4 in serum. The hGCase activity and protein were detected in cells of the liver (massive), lung, and spleen, but not the brain. The visceral tissue storage cells and glucosylceramide (GC) accumulation in hGCase/4L/PS-NA were decreased from that in 4L/PS-NA mice. Turning off hGCase expression with dietary DOX led to reaccumulation of storage cells and of GC in liver, lung, and spleen, and macrophage activation in those tissues. This study demonstrates that conditionally expressed hGCase supplemented the existing mutant mouse GCase to control visceral substrate accumulation in vivo.

Cell cycle dependent intracellular distribution of two spliced isoforms of TCP/ILF3 proteins.

TCP80 is an approximately 80kDa mammalian cytoplasmic protein that binds to a set of mRNAs and inhibits their translation in vitro and ex vivo. This protein has high sequence similarity to interleukin-2 enhancer-binding factors (NF90/ILF3) and the M-phase phosphoprotein (MPP4)/DRBP76. A 110kDa immunologic isoform of TCP80/NF90/MPP4/DRBP76, termed TCP110, also is present in cytoplasm and nuclei of many types of cells. A cDNA sequence coding for TCP110 was derived by 5(')RACE. The TCP110 sequence is identical to ILF3. The gene coding for TCP110/ILF3 mapped to human chromosome 19 and the gene organization was analyzed using TCP80 and TCP110/ILF3 cDNA sequences. The TCP/ILF3 gene spans >34.8kb and contains 21 exons. At least one alternatively spliced product involving exons 19-21 exists and predicts the formation of either TCP80 or TCP110/ILF3. However, the functional relationships of TCP80 and TCP110/ILF3 required elucidation. The metabolic turnover rates and subcellular distribution of TCP80 and TCP110/ILF3 during the cell cycle showed TCP80 to be relatively stable (t(1/2)=5 days) in the cytoplasmic compartment. In comparison, TCP110/ILF3 migrated between the cytoplasmic and nuclear compartments during the cell cycle. The TCP110 C-terminal segment contains an additional nuclear localizing signal that plays a role in its nuclear translocation. This study indicates that the multiple cellular functions, i.e., translation control, interleukin-2 enhancer binding, or cell division, of TCP/ILF3 are fulfilled by alternatively spliced isoforms.