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.

[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.

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.

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.

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.

[Features of uterine contractile function in intrauterine fetal death]. / Osobennosti sokratitel'noi funktsii matki pri vnutriutrobnoi gibeli plodov.

Experiments on 528 pregnant rats studied the specific features of uterine contractility in pre- (7.2%), postimplantation (5.69%), and intrauterine (1.14%) fetal deaths and in ablatio placentae (0.57%) which spontaneously occurred in this group of animals. By applying the multiparametric approach and the methods of the image recognition theory, the authors showed that despite its onset periods, fetal deaths resulted in reduced uterine contractility. It is concluded that the present study provides experimental evidence for the development of weak clinical uterine activity in fetal death in different periods of pregnancy. At the same time, myometrial responses to oxytocin decrease.

[Physico-technological standards of remote neutron radiotherapy and quality assurance (literature review)]. / Fiziko-tekhnicheskie aspekty garantii kachestva neitronnoi distantsionnoi luchevoi terapii i problemy eë obespecheniia (obzor literatury)

Issues in physico-technological support of remote neutron therapy are discussed. On the basis of evaluation of the literature, conclusion is made that a number of important problems are still to be solved.

Zik1, a transcriptional repressor that interacts with the heterogeneous nuclear ribonucleoprotein particle K protein.

The heterogeneous nuclear ribonucleoprotein particle (hnRNP) K protein is comprised of multiple modular domains that serve to engage a diverse group of molecular partners including DNA, RNA, the product of the proto-oncogene vav, and tyrosine and serine/threonine kinases. To identify additional K protein molecular partners and to further understand its function, we used a fragment of K protein as a bait in the yeast two-hybrid screen. The deduced primary structure of one of the positive clones revealed a novel zinc finger protein, hereby denoted as Zik1. In addition to the nine contiguous zinc fingers in the C terminus, Zik1 contains a KRAB-A domain thought to be involved in transcriptional repression. Zik1 and K protein bound in vitro and co-immunoprecipitated from cell extracts indicating that in vivo their interaction is direct. Expression of Gal4 DNA-binding domain-Zik1 fusion protein repressed a gene promoter bearing Gal4-binding elements, indicating that from cognate DNA elements Zik1 is a transcriptional repressor. The known diverse nature of K protein molecular interactions and now the identification of a K protein partner that is a transcriptional repressor lends support to the notion that K protein is a remarkably versatile molecule that may be acting as a docking platform to facilitate communication among molecules involved in signal transduction and gene expression.

ESR and TL dosimetry systems: comparative measurements for human phantom.

Mixtures of small fragments of tooth enamel as well as thermoluminescence (TL) dosimeters were placed into the tissue-equivalent phantom of the human head with skeleton (approximately at the level of the jaws) and irradiated using 137Cs low dose-rate gamma therapeutic sources ('SELEKTRON' LDR 137Cs). Phantom, samples of teeth and TL detectors were irradiated behind water tank to produce scattered irradiation. The same irradiation with the same geometry was performed in air too. For gamma-spectrometry 137Cs sources with very low activity were used but with the same geometry as therapeutic sources. The absorbed dose in enamel was estimated with the help of ESR spectrometer 'ESP-300 E' (Brucker). The samples of tooth enamel were partially used for preliminary dose evaluation by ESR signal before starting of experiment. TL dosimetry was performed by TL reader model 8800 (HARSHAW) using TL dosimeters calibrated with 137Cs. The paper presents data obtained in comparative aspects.

Activation of a nuclear DNA-binding protein recognized by a transcriptional element, bcn-1, from the laminin B2 chain gene promoter.

Treatment of mesangial cells with either phorbol 12-myristate 13-acetate (PMA) or interleukin-1beta induces an increase in laminin B2 chain mRNA levels. In other systems, activation of gene expression by these agents is transcriptionally mediated. To identify transcription factors that control expression of laminin B2 chain gene, we employed a strategy consisting of a computer-based analysis of murine and human gene promoter sequences and gel shift assays. Although overall the laminin B2 chain promoters from the two species have low sequence similarity, the mouse promoter contained sequences that were also contained in one motif, 5'-CCCCGCCCACCTCGCGCGC-3', designated bcn-1, from the human promoter. Treatment of mesangial cells with either PMA or interleukin-1beta induced a transient increase in nuclear DNA binding activity, designated BCN-1, recognized the bcn-1 motif in a gel shift assay. A single nucleotide replacement in the bcn-1 motif abolished DNA binding, indicating that bcn-1.BCN-1 complex formation is highly specific. In transient transfections, the ability of PMA to induce the laminin B2 chain promoter was abolished by mutating the bcn-1 motif. These results suggest that the bcn-1 element and its cognate inducible BCN-1 protein regulate laminin B2 chain gene transcription.

[Study of plant lectins from Viscum album using monoclonal antibodies]. / Issledovanie rastitel'nykh lektinov iz omely beloi s pomoshch'iu monoklonal'nykh antitel.

Monoclonal antibodies (monAT) against both native (TA5, TB12) and denatured (TB33, TB35) plant toxin ML1 from Viscum album have been obtained. The interaction of monAT against native toxin with its isoforms ML2 and ML3 was investigated. It was shown that monAT TA5 to A-chain of ML1 toxin cross-reacted with ML2 and ML3 isoforms. TA5 did not inhibit enzyme activity of A-chain in cell-free rabbit reticulocyte system. It was shown that monAT TB12 reacted with galactose-binding site of B-subunit. Both monAT had no cross-reactions with plant toxin ricin. The binding constants for TA5 with ML1, ML2, ML3 respectively were 4.3.10(7) M-1, 1.2.10(7) M-1, and 0.3.10(7) M-1. The binding constants for TB12 were 2.10(7) M-1 with ML1 toxin, and more than 10(6) M-1 with ML2 and ML3. The nature of heterogeneity in ML toxin family is discussed. Test-systems for ML1 determination in different V. album extracts are suggested.

The state of actin assembly regulates actin and vinculin expression by a feedback loop.

Actin filaments are major determinants of cell shape, motility and adhesion, which control important biological processes including embryonic development and wound healing. These processes are associated with changes in actin assembly, which is regulated by controlling the balance between polymerized and non-polymerized actin. To maintain a significant pool of non-polymerized actin, mechanism(s) linking actin synthesis to its state of polymerization were proposed. We have studied this relationship between actin synthesis and organization by modulating actin assembly using different drugs. Unassembled actin was increased in 3T3 cells using either the Clostridium botulinum C2 toxin, which ADP-ribosylates actin, or by latrunculin A, a Red Sea sponge product, which binds monomeric actin. The synthesis of actin was dramatically reduced in these cells owing to a concomitant decrease in actin RNA level. Similar results were obtained with HeLa cells grown in both monolayer and in suspension, suggesting that cell shape changes associated with drug treatment are not the primary cause for the effect on actin synthesis. In contrast, the scrape-loading of 3T3 cells with phalloidin, a stabilizer of polymerized actin that increased the level of assembled actin, resulted in elevated actin synthesis and RNA content. The expression of vinculin, a major component of adhesion plaques and cell-cell junctions, which is involved in actin-membrane associations, was altered in parallel with that of actin in cells treated with these drugs. The decrease in actin RNA resulted from destabilization of actin mRNA in cells where unassembled actin level was elevated. This is suggested by the unchanged transcription of actin in isolated nuclei from drug-treated cells, and by demonstrating that actin mRNA was degraded faster in cells after C2 toxin treatment than in control cells. This feedback control mechanism is mainly confined to the cytoplasm, as it remained active in enucleated cells. The results suggest the existence of an autoregulatory pathway for the expression of actin and other microfilament-associated proteins which is linked to the state of actin polymerization in the cell.

The interactions of anti-MLI monoclonal antibodies with isoforms of the lectin from Viscum album.

Monoclonal antibodies (mAb) reacting with native (TA5, TB12) and denatured (T33, T35) plant toxin mistletoe lectin I (MLI) from Viscum album have been obtained. The interaction between mAbs and native toxin with ML isoforms (MLII, MLIII) has been investigated. An immunological cross-reaction has been shown to take place for mAb TA5 (anti-A-chain of MLI) between MLII and MLIII isoforms of toxin. TA5 has not inhibited enzyme activity of the A-chain in a rabbit reticulocyte cell-free system. TB12 has been shown to react with the galactose-binding site of the B-chain. TA5 and TB12 have shown no cross-reaction with plant toxin ricin. The association constants for mAbs have been determined. The nature of heterogeneity of the lectins from Viscum album is discussed.

A novel open reading frame in tobacco mosaic virus genome coding for a putative small, positively charged protein.

From sequence comparisons between the tobramovirus genomes an open reading frame (ORF-X) potentially encoding a small, positively charged protein (33- to 45-amino-acids long) was found to overlap the immediate 3' and 5' sides of the transport protein gene and coat protein gene, respectively. In vitro translation of the monocistronic artificial transcripts generated with T7 RNA polymerase yielded a protein of M(r) 4000 (p4) and an unexpected trypsin-sensitive complex of M(r) 54,000 that was resistant to reduction with 2-mercaptoethanol but could be dissociated by 8 M urea. Assembly of this complex was inhibited completely by site-directed mutagenesis within a conserved, positively charged 5-amino-acid long segment of the ORF-X protein. After centrifugation in low salt buffer the 54-kDa complex remained mostly associated with ribosomes. Apparently this complex represents a specific aggregate of the p4 product of ORF-X with a protein of approximate M(r) 50,000 that is a component of the translation apparatus.

[Contact radiotherapy in rheumatoid lesions of the knee joint using Au-198]. / Kontaktnaia luchevaia terapiia pri revmatoidnom porazhenii kolennikh sustavov s ispol'zovaniem 198Au.

An instrumental-calculation method of assessment of an absorbed dose of radiation in the synovial tissue of the knee joint in radionuclide therapy with 198Au using directly measured topometric parameters was used for patients with rheumatoid arthritis. A group of 294 knee joint of 138 patients was considered by way of a retrospective analysis. Dosimetric indices in all cases (except stage IV arthritis) showed values which were not contrary to clinical assessment of the patients: status after therapy.

Stimulation of actin synthesis in phalloidin-treated cells. Evidence for autoregulatory control.

We used the technique of scrape loading to introduce phalloidin into mouse embryo fibroblasts in mass culture. Phalloidin almost completely destroyed actin microfilament bundles, but the amount of polymerized cytoskeleton-associated actin was increased approximately two-fold and the amount of monomeric (Triton X-100 extractable) actin was significantly reduced. The major result of the present study is that the rate of actin synthesis in the phalloidin-treated cells was 2-3 times higher than in the control cells. Northern blot and translation in a cell-free system from rabbit reticulocytes showed that the actin mRNA level significantly increased as a result of phalloidin treatment.