Dynamics of Structural Parameters and Accumulation of Collagen Fibrils in Rat Lung after Inhalations of Surfactant-BL at Various Terms of Bleomycin-Induced Alveolitis.

Rats were subjected to surfactant-BL inhalations at the early and late phases of bleomycininduced alveolitis. In both regimens, the drug reduced the severity of inflammation. In the acute phase of alveolitis, the therapeutic effect of inhalation was accompanied by activation of the synthesis of fine lose collagen fibrils. In the late phase of alveolitis, inhalation of surfactant-BL thickened the fibrils and diminished their population in alveolar walls.

Effectiveness of correction of bleomycin-induced lung injury by early and late administration of surfactant-BL.

Rats were exposed to inhalation of surfactant-BL starting from the first or eighth day after intratracheal administration of bleomycin. At the early stages, the preparation effectively attenuated damage to ultrastructural components of the lung tissue and reduced the severity and extent of subsequent pulmonary pathology.

Therapeutic efficiency of early and late administration of surfactant-BL during bleomycin-induced damage to rat lungs.

Surfactant-BL was administered to rats via the inhalation route from day 1 or day 8 after intratracheal injection of bleomycin. Bronchoalveolar lavage and morphological characteristics of the lungs were compared. Administration of surfactant-BL at the early terms efficiently reduced the severity of bleomycin-induced alveolitis and atelectases.

DNA-hydrolyzing antibodies from sera of autoimmune-prone MRL/MpJ-lpr mice.

Catalytically active antibodies (abzymes) hydrolyzing proteins, polysaccharides, ATP, DNA, and RNA have been detected in the sera of patients with various autoimmune and some viral diseases, but abzymes from the sera of animals are practically unstudied. The development of lupus-like autoimmune disease of MRL/MpJ-lpr mice is an experimental model for study of autoimmune pathologies and immunopathogenesis. In this work, homogeneous IgG preparations were isolated from the sera of MRL/MpJ-lpr mice. These antibodies (Abs), their Fab-fragments, and isolated light chains were shown to possess catalytic activity in DNA hydrolysis, whereas Abs from the sera of control healthy mice did not hydrolyze DNA. The data demonstrate that DNA hydrolyzing activity is an intrinsic property of Abs from MRL/MpJ-lpr mice. It was shown that various markers of autoimmune pathologies (level of total protein concentration in urea (proteinuria), Abs titers to native and denatured DNA, and DNA-hydrolyzing activity of IgG) increased in animals with aging, but they noticeably increased (2-22 times) only after appearance of obvious indicators of pathology independently of age. The highest increase in proteinuria (25-fold), anti-DNA Abs titers (12-19-fold), and abzyme activity (120-fold) was found in mice after their immunization with DNA-protein complex.

Dynamics of antibody nuclease activity in blood of women during pregnancy and lactation.

In human milk we previously found catalytic antibodies (abzymes) catalyzing hydrolysis of DNA, RNA, NMP, NDP, and NTP and also phosphorylation of proteins and lipids. In the present study we have analyzed nuclease activities of antibodies in blood of women during pregnancy and lactation. Blood of healthy male and female volunteers lacked catalytically active antibodies, whereas antibodies from blood of pregnant women hydrolyzed DNA and RNA and their relative activity varied over a wide range. Relative blood abzyme activities significantly increased after delivery and at the beginning of lactation. The highest abzyme activity was observed in blood of parturient women. Although the dynamics of changes in antibody DNase activity during pregnancy was rather individual for each woman, there was a common trend in the increase in antibody activity in the first and/or third trimester of the pregnancy. The DNase activity of IgG and IgM from blood of healthy pregnant women was 4-5 times less than that from pregnant women with pronounced autoimmune thyroiditis.

Juvenile hormone signaling during oogenesis in Drosophila melanogaster.

Juvenile hormone (JH) participates both in the control of insect development and the establishment of reproductive maturity. In cultured Drosophila cells and in ovarian nurse cells, JH and its synthetic analog, methoprene, induce the expression of two related genes. These genes encode highly similar amino acid transport proteins that are homologous to transporters found in a variety of eukaryotes. JhI-21 is a novel Drosophila gene, and minidiscs (mnd) is a gene that was identified earlier. Two JH-inducible genes are regulated by different molecular mechanisms; JhI-21 behaves as a secondary JH-responsive gene, while mnd behaves as a primary responsive gene. Both JhI-21 and mnd transcripts show developmental profiles, which are consistent with JH regulation. Following eclosion, transcripts from JhI-21 and mnd are synthesized in ovarian nurse cells and subsequently sequestered in the mature egg. Their ectopic accumulation in ovaries can be induced by topical methoprene application. In apterous (ap4) mutant adults defective in JH secretion, mnd and JhI-21 RNA levels are severely reduced, but normal abundance is rescued to a high degree by topical methoprene treatment. Based on the evidence, we propose that during sexual maturation of Drosophila, JH provides a signal to the ovary that leads to the production of several maternally inherited mRNAs.

Selective binding of Drosophila BR-C isoforms to a distal regulatory element in the hsp23 promoter.

The Broad-Complex (BR-C) gene plays a key role in the ecdysone regulatory hierarchy. Together with other early ecdysone-inducible genes BR-C transmits the hormonal signal to a set of secondary response genes in a tissue-specific manner. Among its targets is the hsp23 gene. Previously we showed that expression of the hsp23 gene in late third instar is BR-C-dependent, and accompanied by the appearance of a BR-C-dependent DNase I hypersensitive site at position -1400 (DHS-1400). BR-C encodes a family of transcription factors, and we show here that at least three BR-C protein isoforms--Z1, Z2, and Z3--bind to the sequences around DHS-1400 in vitro. A DNase I footprinting assay reveals five protected regions, designated site 1 to site 5, each of which specifically associates with one or several BR-C protein isoforms. We also show that a 100 bp region overlapping site 5, which binds all three isoforms in vitro, is required for hsp23 activity in vivo. The deletion of binding site 5 in a reporter gene construct reproduced the effect of the npr class mutations, that is, hsp23 is no longer expressed in any tissue tested except brain. Thus, BR-C regulates hsp23 expression via direct interaction of the predominant isoform with the distal regulatory element.

The isolation of two juvenile hormone-inducible genes in Drosophila melanogaster.

Juvenile hormone (JH) is an important regulator of both insect development and reproductive maturation. Although the molecular mechanism of JH action is not yet known, there is growing circumstantial evidence that JH directly regulates gene expression. In the absence of a JH target gene, however, this suggestion has remained speculative. Cultured Drosophila S2 cells have been used to identify genes whose expression is regulated by JH. Employing differential display we identified several genes whose transcripts accumulate in cells treated with the JH agonist methoprene. Two of the genes-JhI-1 and JhI-26-were cloned and characterized in detail. For both genes, transcripts showed rapid and specific induction in the presence of either methoprene or JHIII, but not in the presence of other biologically inactive compounds of similar chemical structure. Accumulation of JhI-1 and JhI-26 RNAs requires continuous hormone presence. The developmental expression of the two JH-inducible genes corresponds to the abundance profile of JH in vivo. Furthermore, topical methoprene application to pupae leads to the ectopic accumulation of JhI-1 and JhI-26 transcripts.

Effects of Cr(VI) on the expression of the oxidative stress genes in human lung cells.

Intracellular metabolism of chromium(VI) [Cr(VI)] may lead to oxidative stress and this may account for the ability of Cr(VI) to act as a complete carcinogen. Therefore, we examined the effects of Cr(VI) treatment on the expression of oxidative stress genes in normal human lung LL 24 cells and human lung adenocarcinoma A549 cells. RT-PCR and northern blot analyses were used to determine the steady-state mRNA levels of catalase, glutathione S-transferase, glutathione reductase, Cu/Zn- and Mn-superoxide dismutases, glutathione peroxidase, NAD(P)H:quinone oxidoreductase, heme oxygenase and interleukin 8 in control cells and cells treated with 5-200 microM of Cr(VI). We found that only expression of the heme oxygenase gene is strongly elevated under the treatment with Cr(VI), and only in normal human lung LL 24 cells. Our data showed that even in the absence of Cr(VI) treatment, the level of heme oxygenase gene expression is much higher in A549 cells than in LL 24 cells. As glutathione is believed to play a protective role in cells against different forms of oxidative stress, we studied the correlation between intracellular glutathione levels and the inducibility of the heme oxygenase gene after treatment of cells with Cr(VI). Our results demonstrate that glutathione levels are increased by 35 % of control values in LL 24 cells treated with Cr(VI). The data obtained indicate that heme oxygenase, known to be a stress-inducible gene, may be involved in cellular pathways critical to the carcinogenic activity of Cr(VI) in normal human lung cells. Intracellular glutathione levels and reactive oxygen species do not appear to be primarily responsible for the stress response, induced by Cr(VI) in the studied human cells.

Multiple interactions between hTAFII55 and other TFIID subunits. Requirements for the formation of stable ternary complexes between hTAFII55 and the TATA-binding protein.

We have cloned and characterized the human TATA-binding protein (TBP)-associated factor hTAFII55. hTAFII55, which has no known Drosophila counterpart, is present in both of the previously described TFIIDalpha and TFIIDbeta subpopulations. We describe the interactions of hTAFII55 with other subunits of the transcription factor TFIID. By cotransfection in COS cells, we show that hTAFII55 interacts with hTAFII250, hTAFII100, hTAFII28, hTAFII20, and hTAFII18, but not with hTAFII30 or TBP. Analysis of the binding of hTAFII55 and TBP to hTAFII28 deletion mutants indicates that distinct regions of hTAFII28 are required for these interactions. Although hTAFII55 does not interact by itself with TBP, stable ternary complexes containing hTAFII55 and TBP can be formed in the presence of hTAFII250, hTAFII100, or hTAFII28. These results not only show that hTAFII100 and hTAFII28 interact with TBP, but also that they can nucleate the formation of partial TFIID complexes.

Distinct domains of hTAFII100 are required for functional interaction with transcription factor TFIIF beta (RAP30) and incorporation into the TFIID complex.

TFIID is the DNA binding component of the RNA polymerase II transcriptional machinery and is composed of the TATA binding protein (TBP) and TBP-associated factors (TAFIIs). Here we report the characterization of a new human TAF, hTAFII100, which is the human homologue of Drosophila TAFII80 and yeast TAFII90. hTAFII100 interacts strongly with hTAFII250, hTAFII55 and hTAFII28, less with hTAFII20 and hTAFII18, weakly with TBP and not at all with delta NTAFII135 and hTAFII30. Deletion analysis revealed that the C-terminal half of hTAFII100, which contains six WD-40 repeats, is not required for incorporation into the TFIID complex. Our results suggest that hTAFII100 can be divided into two domains, the N-terminal region responsible for interactions within the TFIID complex and the C-terminal WD repeat-containing half responsible for interactions between hTAFII100 and other factors. An anti-hTAFII100 antibody, raised against a C-terminal epitope, selectively inhibited basal TFIID-dependent in vitro transcription and the specific interaction between hTAFII100 and the 30 kDa subunit of TFIIF (RAP30). We demonstrate that the hTAFII100-TFIIF interaction supports pre-initiation complex formation in the presence of TFIID. Thus, this is the first demonstration that a TAFII functionally interacts with a basal transcription factor in vitro.

Localization of proteins forming the outer surface of isolated metaphase chromosomes.

The outer surface of isolated metaphase chromosomes has been investigated by a method of thermally activated tritium labelling. We show that both chromosomal proteins and DNA are tritium-labelled. Fractionation of the chromosomal proteins reveals that scaffold proteins are the most labelled in condensed and EDTA-decondensed chromosomes. Exposition of some scaffold proteins on the outer surface of metaphase chromosomes is suggested.

Membrane-mediated changes in the structure of chromosomes.

In the present paper the interaction of metaphase chromosomes and chromatin with model and natural lipid membranes was studied. It was shown that chromatin and chromosomes are able to form complexes with membranes in the presence of divalent cations. In such complexes, the typical structure of chromosomes is altered. The character of this alteration in chromosomal structure was investigated with the use of electron microscopy and chemical modification with dimethylsulphate (DMS). The latter is possible because, according to the presented data, the condensation of chromatin into chromosomes is associated with a decrease in accessibility of N-3 in adenine (the protection of the minor groove of DNA) to modifications, and with an increased methylation of N-1 in adenine (the disarrangement of the secondary structure of DNA). It was shown that the interaction of chromosomes with liposomes provides various levels of unfolding up to the appearance of chromatin-like structures. The secondary DNA structure of decondensed chromosomes coincides with the secondary structure of chromosomal but not chromatin DNA, whereas the extent of shielding of the minor groove of DNA in such decondensed structures typical for chromatin DNA. It is possible to suggest that the chromosomal decondensation in telophase of mitosis is initiated by the action of a membrane component of the developing nuclear envelope.

Structure of DNA in metaphase chromosomes of mouse fibroblasts.

We show that N-1 in adenine of chromosomal DNA is methylated by treatment of metaphase chromosomes with dimethylsulphate while this is not the case in chromatin. The data on methylation are consistent with those obtained from the experiments with S1-nuclease treatment of chromatin and chromosomes. This suggests a disarrangement of DNA secondary structure in the metaphase chromosomes.