Cloning of new acylamidase gene from Rhodococcus erythropolis and its expression in Escherichia coli

The gene for new Rhodococcus erythropolis TA37 acylamidase, which possesses unique substrate specificity, has been cloned and expressed in E. coli. Substrates for this enzyme are not only simple amides, such as acetamide and propionamide, but also N-substituted amides, such as 4′-nitroacetanilide. The 1431-bp gene was expressed in E. coli BL21 (DE3) cells on pET16b plasmid under the control of a promoter of the ? 10 gene from the T7 phage. The molecular mass of recombinant acylamidase in E. coli was 55 kDa, which corresponded to that of native acylamidase from Rhodococcus erythropolis TA37. Recombinant acylamidase was able to hydrolize N-substituted amides. A search of a nucleotide database and multiple alignment revealed that acylamidase belonged to the Amidase protein family PF01425, but its nucleotide and amino acid sequences differed significantly from those of the described amidases.     

Mutagen influence with different mechanisms of action on DNA global methylation in human whole-blood lymphocytes in vitro

Data that support the evidence of mutagens known to cause epigenetic abnormalities that could potentially result in genomic instability and the development of cancer rather than to modifications in the human genome at the gene and chromosomal levels only. The level of global methylation in human lymphocytes in vitro caused by exposure to two mutagens with different mechanisms of action, i.e., dioxidine and methyl methanesulphonate (MMS), was demonstrated in the present study. Global methylation was assessed by methyl-sensitive comet assay. An increase in the level of global methylation to 45.64% was revealed during culturing with dioxidine in a concentration of 0.01 mg/mL (p < 0.001), while the addition of dioxidine in a concentration of 0.1 mg/mL resulted in a decreased level of methylation up to 42.31% (p < 0.001). The addition of MMS in concentrations of 0.0025 and 0.01 mg/mL resulted in minor but significant modifications (p < 0.05) of the global methylation level ranged within natural variations in global methylation. Accordingly, the addition of dioxidine in the concentration of 0.1 mg/mL might cause genomic instability and might be considered a potential carcinogen.     

Poriferan mtDNA and animal phylogeny based on mitochondrial gene arrangements

Phylogenetic relationships among the metazoan phyla are the subject of an ongoing controversy. Analysis of mitochondrial gene arrangements is a powerful tool to investigate these relationships; however, its previous application outside of individual animal phyla has been hampered by the lack of informative out-group data. To address this shortcoming, we determined complete mitochondrial DNA sequences for the demosponges Geodia neptuni and Tethya actinia, two representatives of the most basal animal phylum, the Porifera. With sponges as an outgroup, we investigated phylogenetic relationships of nine bilaterian phyla using both breakpoint analysis of global mitochondrial gene arrangements and maximum parsimony analysis of mitochondrial gene adjacencies. Our results provide strong support for a group that includes protostome (but not deuterostome) coelomate, pseudocoelomate, and acoelomate animals, thus clearly rejecting the Coelomata hypothesis. Two other groups of bilaterian animals, Lophotrochozoa and Ambulacraria, are also supported by our analyses. However, due to the remarkable stability of mitochondrial gene arrangements in Deuterostomia and the Ecdysozoa, conclusions on their evolutionary history cannot be drawn.     

Mitochondrial genomes of two demosponges provide insights into an early stage of animal evolution

Mitochondrial DNA (mtDNA) of multicellular animals (Metazoa) is typically a small ( approximately 16 kbp), circular-mapping molecule that encodes 37 tightly packed genes. The structures of mtDNA-encoded transfer RNAs (tRNAs) and ribosomal RNAs (rRNAs) are usually highly unorthodox, and proteins are translated with multiple deviations from the standard genetic code. In contrast, mtDNA of the choanoflagellate Monosiga brevicollis, the closest unicellular relative of animals, is four times larger, contains 1.5 times as many genes, and lacks mentioned peculiarities of animal mtDNA. To investigate the evolutionary transition that led to the specific organization of metazoan mtDNA, we determined complete mitochondrial sequences from the demosponges Geodia neptuni and Tethya actinia, two representatives of the most basal animal phylum, the Porifera. We found that poriferan mtDNAs resemble those of other animals in their compact organization, lack of introns, and a well-conserved animal-like gene order. Yet, they contain several extra genes, encode bacterial-like rRNAs and tRNAs, and use a minimally derived genetic code. Our findings suggest that the evolution of the typical metazoan mtDNA has been a multistep process in which the compact genome organization and the reduced gene content were established prior to the reduction of tRNA and rRNA structures and the introduction of multiple changes of the translation code.     

Eu-heterochromatic rearrangements induce replication of heterochromatic sequences normally underreplicated in polytene chromosomes of Drosophila melanogaster

In polytene chromosomes of D. melanogaster the heterochromatic pericentric regions are underreplicated (underrepresented). In this report, we analyze the effects of eu-heterochromatic rearrangements involving a cluster of the X-linked heterochromatic (Xh) Stellate repeats on the representation of these sequences in salivary gland polytene chromosomes. The discontinuous heterochromatic Stellate cluster contains specific restriction fragments that were mapped along the distal region of Xh. We found that transposition of a fragment of the Stellate cluster into euchromatin resulted in its replication in polytene chromosomes. Interestingly, only the Stellate repeats that remain within the pericentric Xh and are close to a new eu-heterochromatic boundary were replicated, strongly suggesting the existence of a spreading effect exerted by the adjacent euchromatin. Internal rearrangements of the distal Xh did not affect Stellate polytenization. We also demonstrated trans effects exerted by heterochromatic blocks on the replication of the rearranged heterochromatin; replication of transposed Stellate sequences was suppressed by a deletion of Xh and restored by addition of Y heterochromatin. This phenomenon is discussed in light of a possible role of heterochromatic proteins in the process of heterochromatin underrepresentation in polytene chromosomes.     

Activation of NMDA receptors is required for the initiation and maintenance of walking-like activity in the mudpuppy (Necturus Maculatus)

We hypothesized that blocking the activation of N-methyl-D-aspartate (NMDA) receptors prevents the initiation of walking-like activity and abolishes the ongoing rhythmic activity in the spinal cord-forelimb preparation from the mudpuppy. Robust walking-like movements of the limb and rhythmic alternating elbow flexor-extensor EMG pattern characteristic of walking were elicited when continuous perfusion of the spinal cord with solution containing D-glutamate. The frequency of the walking-like activity was dose-dependent on the concentration of D-glutamate in the bath over a range of 0.2 to 0.9 mmol/L. Elevation of potassium concentrations failed to induce walking-like activity. Application of the selective antagonist 2-amino-5-phosphonovalerate (AP-5) produced dose-dependent block of the initiation and maintenance of walking-like activity induced by D-glutamate. Complete block of the activity was achieved when the concentration of AP-5 reached 20 micromol/L. Furthermore, application of L-701,324 (a selective antagonist of the strychnine-insensitive glycine site of NMDA receptor) (1-10 micromol/L) also resulted in complete block of the walking-like activity. In contrast, application of the non-NMDA receptor antagonist 6-cyno-7-nitroquinoxaline-2,3-dione (CNQX) (1-50 micromol/L) induced a dose-dependent inhibition of the burst frequency but failed to result in a complete block. Only at concentration as high as 100 micromol/L, did CNQX cause complete block of the rhythmic activity, presumably through nonspecific action on the strychnine-insensitive glycine site of NMDA receptors. These results suggest that activation of NMDA receptors is required for the initiation and maintenance of walking-like activity. Operation of non-NMDA receptors plays a powerful role in the modulation of the walking-like activity in the mudpuppy.     

In vitro inhibition of the cytopathic action of herpes simplex virus, type 1, with a natural cytokine complex

The antiviral action of a natural cytokine complex (NCC)--the preparation Superlymph and its peptide antimicrobial fraction (AMF)--in the culture of Vero cells infected with type 1 herpes simplex virus (HSV-1), strain VR-3, was studied. The NCC preparation did not alter the morphology of the cells for 6 days and was not toxic for the culture of Vero cells. The NCC and AMF produced a protective antiviral effect, which was manifested by the inhibition of the cytopathic action (CPA) of the virus. In the presence of the preparation, the CPA of HSV-1 was equal to 10(-4.67) ICPD50, while in the control CPA was equal to 10(-5.60). The fraction containing antimicrobial peptides (protegrins) and isolated from NCC, characterized by the method of mass spectrometry, produced the maximum antiviral effect on the cell strain Vero (10(-4.58) ICPD50). Thus Superlymph, an immunomodulator with antiviral activity, could be regarded as an effective preparation for the treatment of HSV infection. The action of such preparation was aimed at the inhibition of the CPA of the virus and the stimulation of the antiviral protective mechanisms of the cell.     

Position-effect variegation in Drosophila melanogaster X chromosome inversion with a breakpoint in a satellite block and its suppression in a secondary rearrangement

In(1LR)pn2a is a pericentric inversion with a euchromatic breakpoint in the 2E polytene region and a heterochromatic breakpoint in the right arm of the X chromosome. It is associated with position-effect variegation (PEV) of the pn, wapl, Pgd and other vital loci of the 2E region, which are relocated near the bulk of the X heterochromatin. Cytological analysis showed that the rearrangement brings the 1A–2E euchromatic segment directly into contact with a major portion of the h34 block, a heterochromatic region that is positively stained by the N-banding technique and contains the AAGAG satellite sequences. Molecular cloning revealed the presence of a new junction between euchromatin and AAGAG satellite sequences and demonstrated that the euchromatic breakpoint of In(1LR)pn2a lies in the vinculin gene. In the X ray-induced secondary rearrangement In(1LR)r30, consisting of a pericentric inversion superimposed on In(1LR)pn2a, the h34 material remains associated with the 2E region but is separated from the rest of the X heterochromatin. In this case, the pn, wapl and Pgd loci no longer variegate, suggesting that the satellite-containing h34 region is not able per se to induce detectable PEV on the adjacent euchromatic genes. Received in revised form: 15 June 1997 / Accepted: 16 September 1997     

Experimental studies of the effect of beaver (<Emphasis Type="Italic">Castor fiber</Emphasis> L.) vital activity products on the formation of zooplankton structure (by the example of growth of two cladoceran species of different sizes)

Experiments in microcosms have demonstrated that beaver vital activity products (BVAPs) promote an increase in concentrations of total nitrogen (N) and total phosphorus (P), a decrease in the N/P value in water, and an increase in the abundance and biomass of bacterioplankton. Under such conditions, the abundance and biomass of small Ceriodaphnia dubia Richard and large Daphnia (Ctenodaphnia) magna Straus, which live separately, increase. The coexistence of these cladocerans in microcosms under the BVAP influences results in a high increase in the abundance and biomass of D. magna; in similar experiments without the influence of BVAP, Ceriodaphnia dubia becomes more abundant. The results of bioassay demonstrate that the number of newborns of Ceriodaphnia dubia decreases in water where Daphnia magna is numerous owing to BVAPs. It is suggested that the vital activity products of large representatives of the genus Daphnia inhibit the fecundity of small species of Cladocera. This fact, along with the high competitiveness of large cladoceran species under conditions of a high level of nutritive base, determine the formation of zooplankton communities in beaver ponds which are characterized by a high abundance and biomass and low uniformity     

Frequent variations in cancer-related genes may play prognostic role in treatment of patients with chronic myeloid leukemia

Genome variability of host genome and cancer cells play critical role in diversity of response to existing therapies and overall success in treating oncological diseases. In chronic myeloid leukemia targeted therapy with tyrosine kinase inhibitors demonstrates high efficacy in most of the patients. However about 15 % of patients demonstrate primary resistance to standard therapy. Whole exome sequencing is a good tool for unbiased search of genetic variations important for prognosis of survival and therapy efficacy in many cancers. We apply this approach to CML patients with optimal response and failure of tyrosine kinase therapy. We analyzed exome variations between optimal responders and failures and found 7 variants in cancer-related genes with different genotypes in two groups of patients. Five of them were found in optimal responders: rs11579366, rs1990236, rs176037, rs10653661, rs3803264 and two in failures: rs3099950, rs9471966. These variants were found in genes associated with cancers (ANKRD35, DNAH9, MAGEC1, TOX3) or participating in cancer-related signaling pathways (THSD1, MORN2, PTCRA). We found gene variants which may become early predictors of the therapy outcome and allow development of new early prognostic tests for estimation of therapy efficacy in CML patients. Normal genetic variation may influence therapy efficacy during targeted treatment of cancers.