[Comparative analysis of enteral feeding practices for prevention of gastrointestinal complications in infants with ductus-dependent congenital heart diseases].

Mesenterial hypoperfusion is one of the main pathogenetic factors of necrotizing enterocolitis (NEC) in infants with ductus-dependent congenital heart diseases. NEC in infants undergoing congenital heart surgery increases mortality and length of hospital stay. NEC is also associated with adverse neurodevelopmental outcome. Optimization of enteral feeding can reduce the risk of gastrointestinal complications. NEC risk factors in infants with congenital heart are of special interest in the literature. This article discusses criteria for enteral feeding initiation and increasing preoperatively and after heart surgery. Enteral feeding protocols of leading cardiac surgical centers are reviewed. Practices to provide high energy and nutrient consumption in infants with congenital heart disease are described.

Differences in contacts of RNA polymerases from Escherichia coli and Thermus aquaticus with lacUV5 promoter are determined by core-enzyme of RNA polymerase.

The interaction of RNA polymerases from Escherichia coli and Thermus aquaticus with lacUV5 promoter was studied at various temperatures. Using DNA-protein cross-linking induced by formaldehyde, it was demonstrated that each RNA polymerase formed a unique pattern of contacts with DNA in the open promoter complex. In the case of E. coli RNA polymerase, beta and sigma subunits were involved into formation of cross-links with the promoter, whereas in the case of T. aquaticus RNA polymerase its beta subunit formed the cross-links with the promoter. A cross-linking pattern in promoter complexes of a hybrid holoenzyme comprised of the core-enzyme of E. coli and sigma subunit of T. aquaticus was similar to that of the E. coli holoenzyme. This suggests that DNA-protein contacts in the promoter complex are primarily determined by the core-enzyme of RNA polymerase. However, temperature-dependent behavior of contact formation is determined by the sigma subunit. Results of the present study indicate that the method of formaldehyde cross-linking can be employed for elucidation of differences in the structure of promoter complexes of RNA polymerases from various bacteria.

[Mutations in beta'-subunit of the Escherichia coli RNA-polymerase influence interaction with downstream duplex DNA in the elongation complex]. / Mutatsii v beta'-sub''edinitse RNK-polimerazy Escherichia coli, vliiaiushchie na vzaimodeistvie s perednim dupleksom DNK v élongatsionnom komplekse.

RNA polymerase (RNAP) exhibits absolute processivity being capable of synthesizing RNA 10(3)-10(5) nucleotides in length without breaking contact with the DNA template. Stability of the elongation complex is thought to depend, in particular, on the RNAP-DNA interactions downstream along the run of transcription. We studied the effects of several deletions and insertions in the RNAP beta'-subunit N-terminal region, which presumably interacts with the downstream duplex DNA in the elongation complex. Most of the mutations obtained led to gross defects in RNAP assembly and disturbed catalytic activity of the enzyme. The mutations reduced stability of both promoter and elongation complexes, probably because they altered the contacts between RNAP and the downstream duplex DNA.

[The RNA polymerase structure-activity studies (1962-2001)]. / Strukturno-funktsional'nye issledovaniia RNK-polimerazy (1962-2001).

The study of RNA polymerase initiated by R.B. Khesin is conducted for about forty years at the laboratory founded by him (since 1989, in collaboration with A. Goldfarb's laboratory). Genetic methods are used in combination with methods of the specific chemical crosslinks of nucleic acids with proteins. The paper assesses the main results of the study in comparison with the X-ray crystallographic data of high resolution obtained recently. A short comparative summary of the RNA polymerase structure has been done for bacteria, archaebacteria, and eukaryotic organelles and nuclei. A brief history of the RNA polymerase study is also presented.

[Horizontal transfer of mercury resistance genes in natural bacterial populations]. / Gorizontal'nyi perenos genov ustoichivosti k soedineniiam rtuti v prirodnykh populiatsiiakh bakterii.

The results of studying the horizontal transfer of mercury resistance determinants in environmental bacterial populations are reviewed. Identical or highly homologous mercury resistance (mer) operons and transposons were found in bacteria of different taxonomic groups from geographically distant regions. Recombinant mer operons and transposons were revealed. The data suggest high frequencies of horizontal transfer and of recombination for mercury resistance determinants. The mechanisms of horizontal gene transfer were elucidated in Gram-negative and Gram-positive bacteria. New transposons were found and analyzed.

[Tn5037-a Tn21-like mercury transposon, detected in Thiobacillus ferrooxidans]. / Tn5037-Tn21-podobnyi rtutnyi transpozon, obnaruzhennyi u Thiobacillus ferrooxidans.

The 6645-bp mercury resistance transposon of the chemolithotrophic bacterium Thiobacillus ferrooxidans was cloned and sequenced. This transposon, named Tn5037, belongs to the Tn21 branch of the Tn21 subgroup, many members of which have been isolated from clinical sources. Having the minimum set of the genes (merRTPA), the mercury resistance operon of Tn5037 is organized similarly to most of the Gram-negative bacteria mer operons and is closest to that of Thiobacillus 3.2. The operator-promoter region of the mer operon of Tn5037 also has the common (Tn21/Tn501-like) structure. However, its inverted, presumably MerR protein binding repeats in the operator/promoter element are two base pairs shorter than in Tn21/Tn501. In the merA region, this transposon shares 77.4, 79.1, 83.2 and 87.8% identical bases with Tn21, Tn501, T. ferrooxidance E-15, and Thiobacillus 3.2, respectively. No inducibility of the Tn5037 mer operon was detected in the in vivo experiments. The transposition system (terminal repeats plus gene tnpA) of Tn5037 was inactive in Escherichia coli K12, in contrast to its resolution system (res site plus gene tnpR). However, transposition of Tn5037 in this host was provided by the tnpA gene of Tn5036, a member of the Tn21 subgroup. Sequence analysis of the Tn5037 res site suggested its recombinant nature.

Probing contacts of phosphate groups of oligonucleotides from the non-template strand of lac UV5 promoter with E. coli RNA polymerase using regioselective cross-linking.

Contacts of phosphate groups at positions -12, -15, and -18 in relation to the transcription initiation site in the non-template strand of lac UV5 promoter with lysines or histidines of E. coli RNA polymerase in the open complex model were studied. A number of synthetic oligonucleotides from the -10-area of the non-template strand containing activated 5'-terminal phosphate group were cross-linked with holo- or core-enzyme of RNA polymerase. 5'-N-Hydroxybenzotriazole phosphodiesters of oligonucleotides were used as phosphate activated derivatives. They are capable of phosphorylating amino groups of lysines and histidines in the enzyme molecule that are brought into proximity with activated phosphate in the complex, resulting in the formation of a covalent bond between the oligonucleotide and the protein. The analysis of the products of cross-linking allowed the protein subunit and the amino acid residue taking part in the formation of the covalent bond for each oligonucleotide to be identified. It was found that all oligonucleotides from the non-template strand of promoter in the complex with the holo-enzyme are bound with the sigma70-subunit. When analyzing the products of partial cleavage of the complexes cross-linked at cysteines and methionines using SDS-PAGE, it was shown that phosphate at position -12 made contacts with His180 or His242 of the sigma70-subunit, the reactive amino acid residue being located between the first and second conservative regions. Phosphate at position -15 is located near lysines from two different areas--between Met413 and Met456 (regions 2.3 and 2.4) and between Met470 and Met507 (region 3.1). Phosphate at position -18 makes preferential contacts with a lysine situated between Met470 and Met507 (region 3.1). Based on the analysis of contacts of phosphate groups and the structure of the isolated sigma70-subunit established previously, a scheme of the mutual arrangement of the oligonucleotide and the sigma70-subunit possessed by the holo-enzyme has been proposed.

[Molecular genetic analysis of the Tn5041 transposition system]. / Molekuliarno-geneticheskii analiz sistemy transpozitsii Tn5041.

A study was made of the transposition of the mercury resistance transposon Tn5041 which, together with the closely related toluene degradation transposon Tn4651, forms a separate group in the Tn3 family. Transposition of Tn5041 was host-dependent: the element transposed in its original host Pseudomonas sp. KHP41 but not in P. aeruginosa PAO-R and Escherichia coli K12. Transposition of Tn5041 in these strains proved to be complemented by the transposase gene (tnpA) of Tn4651. The gene region determining the host dependence of Tn5041 transposition was localized with the use of a series of hybrid (Tn5041 x Tn4651) tnpA genes. Its location in the 5'-terminal one-third of the transposase gene is consistent with the data that this region is involved in the formation of the transposition complex in transposons of the Tn3 family. As in other transposons of this family, transposition of Tn5041 occurred via cointegrate formation, suggesting its replicative mechanism. However, neither of the putative resolution proteins encoded by Tn5041 resolved the cointegrates formed during transposition or an artificial cointegrate in E. coli K12. Similar data were obtained with the mercury resistance transposons isolated from environmental Pseudomonas strains and closely related to Tn5041 (Tn5041 subgroup).

Horizontal spread of mer operons among gram-positive bacteria in natural environments.

Horizontal dissemination of the genes responsible for resistance to toxic pollutants may play a key role in the adaptation of bacterial populations to environmental contaminants. However, the frequency and extent of gene dissemination in natural environments is not known. A natural horizontal spread of two distinct mercury resistance (mer) operon variants, which occurred amongst diverse Bacillus and related species over wide geographical areas, is reported. One mer variant encodes a mercuric reductase with a single N-terminal domain, whilst the other encodes a reductase with a duplicated N-terminal domain. The strains containing the former mer operon types are sensitive to organomercurials, and are most common in the terrestrial mercury-resistant Bacillus populations studied in this work. The strains containing the latter operon types are resistant to organomercurials, and dominate in a Minamata Bay mercury-resistant Bacillus population, previously described in the literature. At least three distinct transposons (related to a class II vancomycin-resistance transposon, Tn1546, from a clinical Enterococcus strain) and conjugative plasmids are implicated as mediators of the spread of these mer operons.

Tn5041: a chimeric mercury resistance transposon closely related to the toluene degradative transposon Tn4651.

This paper reports the discovery and characterization of Tn5041, a novel-type transposon vehicle for dissemination of mercury resistance in natural bacterial populations. Tn5041 (14876 bp), identified in a Pseudomonas strain from a mercury mine, is a Tn3 family mercury resistance transposon far outside the Tn21 subgroup. As in other Tn3 family transposons, Tn5041 duplicates 5 bp of the target sequence following insertion. Tn5041 apparently acquired its mer operon as a single-ended relic of a transposon belonging to the classical mercury resistance transposons of the Tn21 subgroup. The putative transposase and the 47 bp terminal inverted repeats of Tn5041 are closely related to those of the toluene degradative transposon Tn4651 and fall into a distinct subgroup on the fringe of the Tn3 family. The amino acid sequence of the putative resolvase of Tn5041 resembles site-specific recombinases of the integrase family. Besides the mer operon and putative transposition genes, Tn5041 contains a 4 kb region that accommodates a number of apparently defective genes and mobile elements.

[Incorporation of Tn5053 and Tn402 into various plasmids]. / Izuchenie vstraivaniia Tn5053 i Tn402 v razlichnye plazmidy.

Transposons Tn5053 and Tn402 that belong to the novel family of Tn elements are characterized by high selectivity when choosing a target. These transposons integrated with a high frequency into only two of seven large plasmids of various incompatibility groups: RP1 and R446b. The res region of the RP1 plasmid par locus and the res region of the transposon Tn701, included into R446b plasmid, served as targets for both transposons. When Tn701 or par locus integrated into plasmids previously unsuitable for Tn5053 and Tn402 transposition, these plasmids became good targets for both transposons. On the contrary, when the res region of RP1 was damaged impaired, this good target became unsuitable. The insertion sites of Tn5053 and Tn402 were concentrated in the res region of Tn1721 and RP1, but, in some cases, they were at a distance of 100-2000 bp from it.

Four genes, two ends, and a res region are involved in transposition of Tn5053: a paradigm for a novel family of transposons carrying either a mer operon or an integron.

The complete nucleotide sequence of an 8447 bp-long mercury-resistance transposon (Tn5053) has been determined. Tn5053 is composed of two modules: (i) the mercury-resistance module and (ii) the transposition module. The mercury-resistance module carries a mer operon, merRTPFAD, and appears to be a single-ended relic of a transposon closely related to the classical mercury-resistance transposons Tn21 and Tn501. The transposition module of Tn5053 is bounded by 25 bp terminal inverted repeats and contains four genes involved in transposition, i.e. tniA, tniB, tniQ, and tniR. Transposition of Tn5053 occurs via cointegrate formation mediated by the products of the tniABQ genes, followed by site-specific cointegrate resolution. This is catalysed by the product of the tniR gene at the res region, which is located upstream of tniR. The same pathway of transposition is used by Tn402 (Tn5090) which carries the integron of R751. Transposition genes of Tn5053 and Tn402 are interchangeable. Sequence analysis suggests that Tn5053 and Tn402 are representatives of a new family of transposable elements, which fall into a recently recognized super-family of transposons including retroviruses, insertion sequences of the IS3 family, and transposons Tn552 and Tn7. We suggest that the tni genes were involved in the dissemination of integrons.

Molecular characterization of an aberrant mercury resistance transposable element from an environmental Acinetobacter strain.

We present the complete nucleotide sequence of a mer operon located on a 60-kb conjugative plasmid pKLH2 from an environmental bacterium, Acinetobacter calcoaceticus, isolated from a mercury mine. The pKLH2 mer operon has essentially the same gene organization as that of Tn21 and Tn501 from clinical bacteria. The pKLH2 mer operon nucleotide sequence shows 85.5% identity with the Tn501 and 80.9% identity with the Tn21 sequences. Vestigial sequences have been found at the ends of the pKLH2 mer operon, indicating that the pKLH2 mer operon was once a part of a Tn21-like transposon, which had committed suicide by an aberrant resolution event.

Tn5053, a mercury resistance transposon with integron's ends.

We describe a novel type of mercury resistance transposon, Tn5053, which was found in the chromosome of a mercury-resistant Xanthomonas strain isolated from a mercury mine. An 8400 base-pair Tn5053 is bracketed by 25 base-pair inverted repeats that have no sequence homology with inverted repeats of classical mercury resistance transposons Tn501 and Tn21. Instead they show high homology with inverted repeats bracketing the antibiotic resistance segment of Tn21 (integron In2). A 38 base-pair element, which is highly homologous to the inverted repeats of classical mercury resistance transposons has been found within Tn5053 near one of its ends. This internal inverted repeat is fused to the mer operon of Tn5053 in exactly the same way as in the Tn501 mercury resistance transposon. This finding suggests that the mer operon was integrated into the Tn5053 transposition module not through integron-specific pathway but rather via insertion of a classical mercury resistance transposon.

[Plasmid DNA transfection in fibroblasts of athymic rats]. / Issledovanie transfektsii plazmidnoi DNK v fibroblasty bestimusnoi krysy.

Nontumorigenic clone FR-7 cl 13 from fibroblasts of athymic rat was obtained from stroma of human colon carcinoma xenograft propagated on nude animals. Spontaneous transformation of this cells was absent after 40 passages in vitro and treatment with pSV2neo. But cells give rise to tumors in athymic mice after transfection with pEJ. This cell clone can be recommended as cells-targets for transfection.

Protein aggregation and inclusion body formation in Escherichia coli rpoH mutant defective in heat shock protein induction.

Mutations in the rpoH gene, encoding sigma 32, an alternative factor required for transcription of the heat shock genes, result in the extensive aggregation of virtually all cellular proteins and formation of inclusion bodies both under stress and non-stress conditions. Inhibitors of protein synthesis suppress this aggregation, suggesting that newly synthesized proteins preferentially aggregate in rpoH mutants. These data suggest that the heat shock proteins are involved in acquisition of the soluble state (i.e. correct conformation) of the bulk of intracellular proteins after their translation.

[Insertion of (dA-dT)n sequences into the regulatory region of the pho5 gene inhibits its expression]. / Vstraivanie (dA-dT)n-blokov v reguliatornuiu oblast' gena pho5 narushaet ego ékspressiiu.

We have studied the effect of some regular sequences namely (dA-dT)n, (dA)n and (dG-dC)n on the Saccharomyces cerevisiae PHO5 gene expression. These sequences were inserted into the ClaI site, located between two phosphate-responsible upstream activating sequences (UAS's). A modified PHO5 gene cloned in vector plasmids was used to transform the pho3, pho5 yeast strain and the level of acid phosphatase expression in various conditions was measured. We show that the insertion of (dA-dT)n blocks, but not (dA)n or (dG-dC)n, leads to the dramatic decrease of PHO5 gene expression in a derepressed state. (dA-dT)n inserts also inhibit the expression of PHO5 gene which was put under the control of heterologous UASgal.

Heat shock response in Escherichia coli promotes assembly of plasmid encoded RNA polymerase beta-subunit into RNA polymerase.

Escherichia coli cells, carrying a rifampicin sensitive RNA polymerase beta-subunit gene in the chromosome and a rifampicin resistant beta-subunit gene placed under the control of a strong promoter in a multicopy plasmid, are unable to grow in the presence of rifampicin, despite the accumulation of large quantities of the resistant subunit. A major portion of the overproduced subunit is found in an insoluble form. Conditions known to induce the heat shock proteins (hsps), e.g. elevated temperature or the presence of ethanol in the growth medium, increase the amount of the plasmid-borne beta-subunit which apparently assembles into active RNA polymerase and makes the plasmid bearing cells rifampicin resistant. Alternatively, plasmid-borne subunits assemble into RNA polymerase with low efficiency in rpoH mutant cells known to have reduced level of hsps. We suggest that the plasmid-borne subunit is poorly assembled into RNA polymerase and that hsps promote the assembly by interfering with beta-subunit aggregation.