[ORGANIZED PNEUMONIA IN A PATIENT WITH ULCERATIVE COLITIS].

The article presents a case of organized pneumonia development in 44-year-old patient with ulcerative colitis, successfully permitted on the background of treatment with high doses of systemic steroids. The authors consider the case as extra-intestinal IBD-associated pulmonary disease.

[Indexes of immunity and local protection in humans with intestine dysbacteriosis].

AIM: Study indexes of immunity and local protection in humans with intestine dysbacteriosis. MATERIALS AND METHODS: Qualitative and quantitative intestine microbiocenosis, content of gamma-interferon (EIA method) in coprofiltrates in 204 individuals were studied, data from immunograms of 123 individuals with bacteriologically confirmed dysbacteriosis were analyzed. RESULTS: The presence of immune deficiency mainly by T-cell type was established in 92.7+/-2.4% of individuals with intestine dysbacteriosis. Significant variations in -interferon content in coprofiltrates of examined individuals was detected (from no less than 5 pcg/ml to 240 pcg/ml), a statistically significant dependence of gamma-interferon content in coprofiltrates on the number of opportunistic microbes and atypical escherichia (including hemolytic) in intestine microbiocenosis was determined. CONCLUSION: The presence of T-cell type immune deficiency in individuals with intestine dysbacteriosis combined with a reduced local protection, and the content of gamma-interferon in coprofiltrates gives evidence not only on the reduction of local protection but also to some extent mirrors the degree of this reduction.

Chromatin structure of two genomic sites for targeted transgene integration in induced pluripotent stem cells and hematopoietic stem cells.

Achieving transgene integration into preselected genomic sites is currently one of the central tasks in stem cell gene therapy. A strategy to mediate such targeted integration involves site-specific endonucleases. Two genomic sites within the MBS85 and chemokine (C-C motif) receptor 5 (CCR5) genes (AAVS1 and CCR5 zinc-finger nuclease (CCR5-ZFN) sites, respectively) have recently been suggested as potential target regions for integration as their disruption has no functional consequence. We hypothesized that efficient transgene integration maybe affected by DNA accessibility of endonucleases and therefore studied the transcriptional and chromatin status of the AAVS1 and CCR5 sites in eight human induced pluripotent stem (iPS) cell lines and pooled CD34+ hematopoietic stem cells (HSCs). Matrix chromatin immunoprecipitation (ChIP) assays demonstrated that the CCR5 site and surrounding regions possessed a predominantly closed chromatin configuration consistent with its transcriptional inactivity in these cell types. In contrast, the AAVS1 site was located within a transcriptionally active region and exhibited an open chromatin configuration in both iPS cells and HSCs. To show that the AAVS1 site is readily amendable to genome modification, we expressed Rep78, an AAV2-derived protein with AAVS1-specific endonuclease activity, in iPS cells after adenoviral gene transfer. We showed that Rep78 efficiently associated with the AAVS1 site and triggered genome modifications within this site. On the other hand, binding to and modification of the CCR5-ZFN site by a ZFN was relatively inefficient. Our data suggest a critical influence of chromatin structure on efficacy of site-specific endonucleases used for genome editing.

Altered adipocyte structure and function in nutritionally programmed microswine offspring.

Adipose tissue (AT) dysfunction links obesity of any cause with cardiometabolic disease, but whether early-life nutritional deficiency can program adipocyte dysfunction independently of obesity is untested. In 3-5-month-old juvenile microswine offspring exposed to isocaloric perinatal maternal protein restriction (MPR) and exhibiting accelerated prepubertal fat accrual without obesity, we assessed markers of acquired obesity: adiponectin and tumor necrosis factor (TNF)-α messenger ribonucleic acid (mRNA) levels and adipocyte size in intra-abdominal (ABD-AT) and subcutaneous (SC-AT) adipose tissues. Plasma cortisol, leptin and insulin levels were measured in fetal, neonatal and juvenile offspring. In juvenile low-protein offspring (LPO), adipocyte size in ABD-AT was reduced 22% (P = 0.011 v. controls), whereas adipocyte size in SC-AT was increased in female LPO (P = 0.05) and normal in male LPO; yet, adiponectin mRNA in LPO was low in both sexes and in both depots (P < 0.001). Plasma leptin (P = 0.004) and cortisol (P < 0.05) were reduced only in neonatal LPO during MPR. In juveniles, correlations between % body fat and adiponectin mRNA, TNF-α mRNA or plasma leptin were significant in normal-protein offspring (NPO) but absent in LPO. Plasma glucose in juvenile LPO was increased in males but decreased in females (interaction, P = 0.023); plasma insulin levels and insulin sensitivity were unaffected. Findings support nutritional programming of adipocyte size and gene expression and subtly altered glucose homeostasis. Reduced adiponectin mRNA and adipokine dysregulation in juvenile LPO following accelerated growth occurred independently of obesity, adipocyte hypertrophy or inflammatory markers; thus, perinatal MPR and/or growth acceleration can alter adipocyte structure and disturb adipokine homeostasis in metabolically adverse patterns predictive of enhanced disease risk.

Maternal malnutrition and placental insufficiency induce global downregulation of gene expression in fetal kidneys.

Malnutrition during pregnancy causes intrauterine growth restriction and long-term changes in the offspring's physiology and metabolism. To explore molecular mechanisms by which the intrauterine environment conveys programming in fetal kidneys, an organ known to undergo substantial changes in many animal models of late gestational undernutrition, we used a microswine model of maternal protein restriction (MPR) in which sows were exposed to isocaloric low protein (LP) diet during late gestation/early lactation to encompass the bulk of nephrogenesis. To define general v. model-specific effects, we also used a sheep model of placental insufficiency. In kidneys from near-term fetal and neonatal microswine LP offspring, per cell levels of total RNA, poly(A)+ mRNA and transcripts of several randomly chosen housekeeping genes were significantly reduced compared to controls. Microarray analysis revealed only a few MPR-resistant genes that escape such downregulation. The ratio of histone modifications H3K4m3/H3K9m3 (active/silenced) was reduced at promoters of downregulated but not MPR-resistant genes suggesting that transcriptional suppression is the point of control. In juvenile offspring, on a normal diet from weaning, cellular RNA levels and histone mark patterns were recovered to near control levels, indicating that global repression of transcription is dependent on ongoing MPR. Importantly, cellular RNA content was also reduced in ovine fetal kidneys during placental insufficiency. These studies show that global repression of transcription may be a universal consequence of a poor intrauterine environment that contributes to fetal restriction.

[Features of microbiocenosis composition of large intestine of humans with various degree of local metabolic disorders].

AIM: Detection of features of qualitative and quantitative composition of large intestine microbiocenosis of humans with various degree of local metabolic disorders during dysbacterioses due to various causes. MATERIALS AND METHODS: Microflora of large intestine and content of malonic dialdehyde (MDA) in coprofiltrates of 330 adult humans with large intestine dysbacterioses due to various causes were studied. RESULTS: It was established, that high MDA content in coprofiltrates matches higher quantities of opportunistic microorganisms and atypical escherichia in microflora composition of large intestine. Relation of MDA composition in coprofiltrates and factors that cause dysbacteriosis were not detected. CONCLUSION: The studies performed give evidence that changes in local metabolic activity may be a single mechanism of development of large intestine dysbacterioses that are caused by various factors.

[Dynamic health state control in personnel of the Radiology Department of Bryansk Regional Oncological Dispensary].

The health state of personnel of the Radiology Department of Bryansk Regional Oncological Dispensary before and after Chernobyl Nuclear Disaster is analyzed using an automated classifying system. The system operation is based on analysis of peripheral blood count.

Interaction between human alpha2-macroglobulin and duodenase, a serine proteinase with dual specificity.

Interaction between a serine proteinase from bovine duodenum and human serum alpha(2)-macroglobulin (alpha(2)-MG) was studied. alpha(2)-MG is established to be one of the most effective duodenase inhibitors. The enzyme is completely inhibited in less than 30 sec at equimolar ratio of the inhibitor and enzyme (concentration 2 x 10(-8) M). Under identical conditions, the rate of duodenase association with alpha(2)-MG is at least 2.5-fold higher than the rate of chymotrypsin association with this inhibitor. The interaction with duodenase results in proteolysis of the inhibitor subunit in the "bait region". Similarly to other proteases, duodenase in the complex with alpha(2)-MG retains the intact catalytic apparatus and ability to hydrolyze some small substrates. But the duodenase-inhibitor complex is fully inactive to proteins (bovine serum albumin). The stoichiometry of the enzyme interaction with the inhibitor is 2 : 1 (mol/mol). Based on the association rate constant and the termination time of the duodenase and alpha(2)-MG in vivo association, alpha(2)-MG is suggested to be a physiological regulator of the enzyme.

Editing of hnRNP K protein mRNA in colorectal adenocarcinoma and surrounding mucosa.

The heterogeneous nuclear ribonucleoprotein K (hnRNP K) protein is an RNA-binding protein involved in many processes that compose gene expression. K protein is upregulated in the malignant processes and has been shown to modulate the expression of genes involved in mitogenic responses and tumorigenesis. To explore the possibility that there are alternative isoforms of K protein expressed in colon cancer, we amplified and sequenced K protein mRNA that was isolated from colorectal cancers as well as from normal tissues surrounding the tumours. Sequencing revealed a single G-to-A base substitution at position 274 that was found in tumours and surrounding mucosa, but not in individuals that had no colorectal tumour. This substitution most likely reflects an RNA editing event because it was not found in the corresponding genomic DNAs. Sequencing of RNA from normal colonic mucosa of patients with prior resection of colorectal cancer revealed only the wild-type K protein transcript, indicating that G274A isoform is tumour related. To our knowledge, this is the first example of an RNA editing event in cancer and its surrounding tissue, a finding that may offer a new diagnostic and treatment marker.

Insulin alters heterogeneous nuclear ribonucleoprotein K protein binding to DNA and RNA.

The interaction of the multimodular heterogeneous nuclear ribonucleoprotein (hnRNP) K protein with many of its protein and nucleic acid partners is regulated by extracellular signals. Acting as a docking platform, K protein could link signal-transduction pathways to DNA- and RNA-directed processes such as transcription, mRNA processing, transport, and translation. Treatment of hepatocyte culture with insulin increased K protein tyrosine phosphorylation. Insulin altered K protein interaction with RNA and DNA in vitro. Administration of insulin into mice had similar effects on K protein in liver. Coimmunoprecipitations of RNA with K protein revealed preferential in vivo K protein binding of a subset of transcripts, including the insulin-inducible c-fos mRNA. These results suggest a class of insulin pathways that signal nucleic acid-directed processes that involve K protein.

Role of tyrosine phosphorylation in the regulation of the interaction of heterogenous nuclear ribonucleoprotein K protein with its protein and RNA partners.

The heterogeneous nuclear ribonucleoprotein K protein recruits a diversity of molecular partners and may act as a docking platform involved in such processes as transcription, RNA processing, and translation. We show that K protein is tyrosine-phosphorylated in vitro by Src and Lck. Treatment with H(2)O(2)/Na(3)VO(4), which induces oxidative stress, stimulated tyrosine phosphorylation of K protein in cultured cells and in intact livers. Tyrosine phosphorylation increased binding of Lck and the proto-oncoprotein Vav to K protein in vitro. Oxidative stress increased the association of K protein with Lck and Vav, suggesting that tyrosine phosphorylation regulates the ability of K protein to recruit these effectors in vivo. Translation-based assay showed that K protein is constitutively bound to many mRNAs in vivo. Native immunoprecipitated K protein-mRNA complexes were disrupted by tyrosine phosphorylation, suggesting that the in vivo binding of K protein to mRNA may be responsive to the extracellular signals that activate tyrosine kinases. This study shows that tyrosine phosphorylation of K protein regulates K protein-protein and K protein-RNA interactions. These data are consistent with a model in which functional interaction of K protein is responsive to changes in the extracellular environment. Acting as a docking platform, K protein may bridge signal transduction pathways to sites of nucleic acid-dependent process such as transcription, RNA processing, and translation.

[Innervation of the humerus metaepiphysis in newborn and one year old children]. / Innervatsiia metaépifiza plechevoi kosti novorozhdennykh i detei pervogo goda zhizni.

Analysis of home and foreign literature on metaepiphyseal cartilage innervation and blood supply was presented, data on these organ diseases substantiate theoretical and clinical significance of the study was performed. Using macro-microscopic preparation, histological study, automatic image analyzer branches of humerus proximal metaepiphysis were shown to originate from axillary and subscapulary nerves. Nerves and vessels penetrating in the cartilaginous canals form neurovascular complexes. Cartilaginous canals are located unevenly. Nervous structures were found in the canals and their characteristics was given.

Regulated interaction of protein kinase Cdelta with the heterogeneous nuclear ribonucleoprotein K protein.

The heterogeneous nuclear ribonucleoprotein (hnRNP) K protein recruits a diversity of molecular partners that are involved in signal transduction, transcription, RNA processing, and translation. K protein is phosphorylated in vivo and in vitro by inducible kinase(s) and contains several potential sites for protein kinase C (PKC) phosphorylation. In this study we show that K protein is phosphorylated in vitro by PKCdelta and by other PKCs. Deletion analysis and site-directed mutagenesis revealed that Ser302 is a major K protein site phosphorylated by PKCdelta in vitro. This residue is located in the middle of a short amino acid fragment that divides the two clusters of SH3-binding domains. Mutation of Ser302 decreased the level of phosphorylation of exogenously expressed K protein in phorbol 12-myristate 13-acetate-treated COS cells, suggesting that Ser302 is also a site for PKC-mediated phosphorylation in vivo. In vitro, PKCdelta binds K protein via the highly interactive KI domain, an interaction that is blocked by poly(C) RNA. Mutation of Ser302 did not alter the K protein-PKCdelta interaction in vitro, suggesting that phosphorylation of this residue alone is not sufficient to alter this interaction. Instead, binding of PKCdelta to K protein in vitro and in vivo was greatly increased by K protein phosphorylation on tyrosine residues. The ability of PKCdelta to bind and phosphorylate K protein may serve not only to alter the activity of K protein itself, but K protein may also bridge PKCdelta to other K protein molecular partners and thus facilitate molecular cross-talk. The regulated nature of the PKCdelta-K protein interaction may serve to meet cellular needs at sites of active transcription, RNA processing and translation in response to changing extracellular environment.

Inducible transcriptional activity of bcn-1 element from laminin gamma1-chain gene promoter in renal and nonrenal cells.

Laminin is a major component of the extracellular matrix whose expression is regulated by growth factors. The laminin gamma1-chain promoter contains a newly identified transcriptional element denoted bcn-1 that is both active and inducible in mesangial cells. In this study, we explored activation of the bcn-1 element in other renal and nonrenal cells. Treatment of rat glomerular epithelial cells (GEC) with phorbol 12-myristate 13-acetate (PMA) increased activity of the bcn-1 transcriptional element, within the context of the native laminin gamma1-chain promoter or when cloned upstream of a heterologous promoter. Treatment of GEC with PMA induced nuclear DNA-binding activity, BCN-1, which was recognized by the bcn-1 motif in a gel shift assay. These results provide evidence that the bcn-1 motif and its cognate BCN-1 factor(s) may regulate transcription of the laminin gamma1-chain in GEC. The bcn-1 element and its cognate BCN-1 DNA-binding activity were also inducible in monkey kidney COS-7 and in human T cell Jurkat lines. SDS-PAGE of in situ ultraviolet cross-linked nucleoproteins from GEC, COS, and Jurkat cells revealed one major 110-115 kDa adduct in all three cell lines. These results demonstrate that the bcn-1 element is active in renal and nonrenal cells from different mammalian species where the same protein contributes to the inducible BCN-1 DNA-binding activity.

Point mutations in the WD40 domain of Eed block its interaction with Ezh2.

The Polycomb group proteins are involved in maintenance of the silenced state of several developmentally regulated genes. These proteins form large aggregates with different subunit compositions. To explore the nature of these complexes and their function, we used the full-length Eed (embryonic ectoderm development) protein, a mammalian homolog of the Drosophila Polycomb group protein Esc, as a bait in the yeast two-hybrid screen. Several strongly interacting cDNA clones were isolated. The cloned cDNAs all encoded the 150- to 200-amino-acid N-terminal fragment of the mammalian homolog of the Drosophila Enhancer of zeste [E(z)] protein, Ezh2. The full-length Ezh2 bound strongly to Eed in vitro, and Eed coimmunoprecipitated with Ezh2 from murine 70Z/3 cell extracts, confirming the interaction between these proteins observed in yeast. Mutations T1031A and T1040C in one of the WD40 repeats of Eed, which account for the hypomorphic and lethal phenotype of eed in mouse development, blocked binding of Ezh2 to Eed in a two-hybrid interaction in yeast and in mammalian cells. These mutations also blocked the interaction between these proteins in vitro. In mammalian cells, the Gal4-Eed fusion protein represses the activity of a promoter bearing Gal4 DNA elements. The N-terminal fragment of the Ezh2 protein abolished the transcriptional repressor activity of Gal4-Eed protein when they were coexpressed in mammalian cells. Eed and Ezh2 were also found to bind RNA in vitro, and RNA altered the interaction between these proteins. These findings suggest that Polycomb group proteins Eed and Ezh2 functionally interact in mammalian cells, an interaction that is mediated by the WD40-containing domain of Eed protein.

Cloning of rat laminin gamma 1-chain gene promoter reveals motifs for recognition of multiple transcription factors.

We have previously shown that laminin gamma 1 (laminin B2)-chain mRNA levels increase in response to treatment of rat glomerular epithelial cells (GEC) with the cytokine interleukin-1 beta (IL-1 beta) [C. A. Richardson, K. L. Gordon, W. G. Couser, and K. Bomsztyk. Am. J. Physiol. 268 (Renal Fluid Electrolyte Physiol. 37): F273-F278, 1995]. IL-1 beta-induced increase in laminin gamma 1-chain gene expression is likely to be transcriptionally regulated. As the laminin gamma 1-chain gene promoter had not previously been cloned in the rat, we cloned the 5'-flanking region of this gene from a rat genomic library. Like the human and murine laminin gamma 1-chain gene promoters, the rat laminin gamma 1-chain gene fragment spanning from nucleotides -1104 to +109, relative to the start codon, is "GC" rich and lacks TATA or CAAT boxes. This rat laminin gamma 1-chain gene promoter region appears to contain at least two transcription initiation sites, i.e., position -169 and -234. In transient transfections in GEC, the -1104/+35 and -1104/-15 fragments cloned upstream of a luciferase reporter gene had very little constitutive activity and were not IL-1 beta responsive. In sharp contrast, a -1104/-234 fragment exhibited constitutive activity and was IL-1 beta responsive. The -1104/-234 fragment contains motifs that recognize Sp1, BCN-1, and ApoE-B1-AP1 DNA-binding activities in GEC nuclear extracts. Collectively, the results of this study suggest that multiple inducible transcription factors may regulate laminin gamma 1-chain gene promoter activity in GEC.

The product of the murine homolog of the Drosophila extra sex combs gene displays transcriptional repressor activity.

The heterogeneous nuclear ribonucleoprotein K protein represents a novel class of proteins that may act as docking platforms that orchestrate cross-talk among molecules involved in signal transduction and gene expression. Using a fragment of K protein as bait in the yeast two-hybrid screen, we isolated a cDNA that encodes a protein whose primary structure has extensive similarity to the Drosophila melanogaster extra sex combs (esc) gene product, Esc, a putative silencer of homeotic genes. The cDNA that we isolated is identical to the cDNA of the recently positionally cloned mouse embryonic ectoderm development gene, eed. Like Esc, Eed contains six WD-40 repeats in the C-terminal half of the protein and is thought to repress homeotic gene expression during mouse embryogenesis. Eed binds to K protein through a domain in its N terminus, but interestingly, this domain is not found in the Drosophila Esc. Gal4-Eed fusion protein represses transcription of a reporter gene driven by a promoter that contains Gal4-binding DNA elements. Eed also represses transcription when recruited to a target promoter by Gal4-K protein. Point mutations within the eed gene that are responsible for severe embryonic development abnormalities abolished the transcriptional repressor activity of Eed. Results of this study suggest that Eed-restricted homeotic gene expression during embryogenesis reflects the action of Eed as a transcriptional repressor. The Eed-mediated transcriptional effects are likely to reflect the interaction of Eed with multiple molecular partners, including K protein.

The beet yellows closterovirus p65 homologue of HSP70 chaperones has ATPase activity associated with its conserved N-terminal domain but does not interact with unfolded protein chains.

The positive-strand RNA genome of beet yellows closterovirus (BYV) encodes a 65 kDa protein (p65) related to the HSP70 family of cell chaperones. The full-sized BYV p65, and N- and C-terminal fragments, with (His)6 tails, were overexpressed in bacteria and purified by metal-chelate chromatography. Using a polyclonal antiserum raised against the C-terminal fragment of p65, evidence was obtained for expression of the viral protein in planta. Purified recombinant p65 and its N-terminal 40 kDa fragment exhibited Mg2+-dependent ATPase activity in vitro. However, unlike its cellular HSP70 homologues, p65 was unable to bind to denatured protein and its ATPase activity was not stimulated by synthetic peptides which are known to stimulate HSP70 ATPases. Hence, the BYV p65, although being a chaperone-type ATPase, may have a distinct substrate specificity and function in BYV-infected cells.