[Low-molecular DNA in blood plasma and spinal fluid of cancer patients].

Enhanced preoperative levels of low-molecular DNA in blood plasma and in cerebrospinal fluid were established in cancer patients. They rose even higher after surgery due to stress. Possible mechanisms are discussed.

Modulation of equine articular chondrocyte messenger RNA levels following brief exposures to recombinant equine interleukin-1beta.

The effect of recombinant equine IL-1beta (EqIL-1beta) on steady-state mRNA levels of equine articular chondrocytes in high-density monolayer culture was investigated using a customized cDNA array analysis. Total RNA samples isolated from chondrocytes cultured in media alone or with the addition of 1 ng/ml EqIL-1beta for 1-, 3-, and 6-h durations of exposure were reverse transcribed, radiolabeled, and hybridized to a customized 380-target cDNA array. Means of duplicate log base 2 transformed hybridization signals were normalized to equine glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mean signal intensities. Differentially expressed transcripts were identified using a two-stage mixed linear analysis of variance model (Statistical Analysis Software, Cary, NC). A time-dependent pattern was observed in the number of transcripts increased > or =two-fold in response to EqIL-1beta after 1, 3 and 6h (1, 2 and 109 transcripts, respectively). At 6 h of EqIL-1beta stimulation, signal intensities for 88 cDNA targets with purported function in processes related to cell cycle, intracellular signaling, transcription, translation, extracellular matrix turnover, and inflammation, as well as a number of cDNAs lacking homology to previously reported cDNA sequences, were increased >two-fold and were associated with p<0.05. Principal component analysis identified a vector component ( approximately 10% of the total variation) corresponding to a potential EqIL-1beta co-regulation of cell cycle associated gene transcription. These results support and expand our existing comprehension of the complex role of IL-1 in modulated chondrocyte gene expression and suggest the involvement of specific target gene up-regulation and activation of downstream inflammatory cascade mediators. This study adds to the current understanding of the molecular events associated with an IL-1 induced inflammation and pathobiologic processes that may be associated with the development of equine osteoarthritis.

Reduced birth defects caused by maternal immune stimulation in diabetic ICR mice: lack of correlation with placental gene expression.

Diabetes mellitus alters placental structure and function, events that may be related to embryopathy. Three different methods of maternal immune stimulation that modulate placental function and that result in approximately equal reduction of diabetic embryopathy were studied: footpad injection with complete Freund's adjuvant, intraperitoneal injection with granulocyte-macrophage colony stimulating factor (GM-CSF), or intraperitoneal injection with interferon-gamma (IFN-gamma). A gene microarray was then used to examine expression of 151 placental genes. We hypothesized that maternal immune stimulation may overcome an embryopathy-inducing effect of diabetes on placenta, that might be detected by a shared profile of placental gene expression changes induced by the different immune stimulation procedures. However, the immune stimulation that caused the greatest reduction in birth defect incidence, IFN-gamma, did not change the placental gene expression profile as compared to control or diabetes. Complete Freund's adjuvant and GM-CSF significantly changed placental gene expression relative to control or diabetes, but differentially affected such genes. No common pattern of improved cytokine, cell-cycle, apoptotic, transcription factor, or other gene expression was identified, that might explain the ability of this procedure to reduce birth defects. These data suggest that maternal immune stimulation reduces birth defects in diabetic mice by a mechanism independent of placenta.

Reduced birth defects caused by maternal immune stimulation may involve increased expression of growth promoting genes and cytokine GM-CSF in the spleen of diabetic ICR mice.

Maternal immune stimulation in mice decreases fetal abnormalities caused by diverse etiologies. Growth factors produced by activated immune cells were proposed to be key mediators that may exert their effects on placenta or embryo. Diabetes disrupts the secretion of cytokines, which may associate with diabetic embryopathy. Three different methods of maternal immune stimulation that result in approximately equal reduction of diabetic embryopathy were used in the present studies: footpad injection with complete Freund's adjuvant (CFA), intraperitoneal (i.p.) injection with granulocyte-macrophage colony-stimulating factor (GM-CSF), or i.p. injection with interferon-gamma (IFN-gamma). A gene microarray was then used to examine expression of a selected gene panel in splenic leukocytes. We hypothesized that maternal immune stimulation may act by overcoming altered gene expression patterns of immune cells in the diabetic mice, which partially mitigates the teratogenic effect of diabetes. It further seemed likely that a shared profile of splenic gene expression changes induced by the different immune stimulation procedures may be identified and related to reduced teratogenesis. The three procedures produced a common altered gene expression profile. Significantly affected genes included apoptotic and anti-apoptotic genes, and genes controlling cellular proliferation, and likely reflect a state of immune activation. The GM-CSF gene was up-regulated by all three immune stimulation procedures. The protein product of this gene regulates placental development, and was recently associated with reduced cleft palate in immune-stimulated pregnant mice after exposure to urethane. These data suggest that further studies of GM-CSF as mediator of reduced birth defects in teratogen-challenged, immune-stimulated mice are warranted.

Maternal immune stimulation reduces both placental morphologic damage and down-regulated placental growth-factor and cell cycle gene expression caused by urethane: are these events related to reduced teratogenesis?

Activation of the maternal immune system in mice decreased cleft palate caused by the chemical teratogen, urethane. Direct and indirect mechanisms for this phenomenon have been suggested, including maternal macrophages that cross the placenta to find and eliminate pre-teratogenic cells, or maternal immune proteins (cytokines) that cross placenta to alleviate or partially alleviate toxicant-mediated effects in the developing fetus. A third mechanism to explain improved fetal developmental outcome in teratogen-challenged pregnant mice might involve beneficial effects of immune stimulation on the placenta. In the present experiments, urethane treatment altered placental morphology and impaired placental function, the latter indicated by down-regulated activity of cell cycle genes and of genes encoding cytokines and growth factors. Maternal immune stimulation with either Freund's complete adjuvant (FCA) or interferon-gamma (IFNgamma) reduced morphologic damage to the placenta caused by urethane and normalized expression of several genes that were down-regulated by urethane. Urethane treatment also shifted placental cytokine gene expression toward a T cell helper 1 (Th1) profile, while immunostimulation tended to restore a Th2 profile that may be more beneficial to pregnancy and fetal development. These data suggest that the beneficial effects of maternal immune stimulation on fetal development in teratogen-exposed mice may, in part, result from improved placental structure and function.

Immune stimulation in urethane-exposed pregnant mice increases expression level of spleen leukocyte genes for TGFbeta3 GM-CSF and other cytokines that may play a role in reduced chemical-induced birth defects.

For unknown reasons, activation of the maternal immune system in mice reduces morphologic defects caused by diverse teratogenic agents. Such immune stimulation of the maternal animal has been correlated with altered cytokine mRNA transcripts in the placenta (e.g., TGFbeta2) as well as in fetal target tissues of the teratogen (e.g., TNFalpha in fetal heads of cyclophosphamide-exposed pregnant mice). The teratogen urethane was reported to down-regulate cell cycle and apoptotic regulatory genes in fetal mouse heads that displayed cleft palate, an effect that was also reversed by maternal immune stimulation. The molecular mediators of the above phenomena have not been identified, however proteins synthesized and released by activated maternal immune cells have been suggested. The present studies therefore evaluated the effects of maternal immune stimulation in urethane-exposed mice on thymus and spleen leukocyte populations, in an attempt to identify events that may correlate with protection against birth defects. Immune stimulation did not change the hypocellularity of the thymus nor the altered T cell differentiation caused by urethane. A limited and transient increase in splenic leukocyte number, including increased T and B lymphocytes and macrophages, was caused by immune stimulation and was not felt to play a significant role in reduced morphologic defects. Urethane treatment caused down-regulated expression of numerous genes involved in cell-cycle control, while maternal immune stimulation caused comparative up-regulation of many of these genes. Coordinate shifts in gene expression by treatment were evaluated using principal component analysis, which identified several growth factor genes that were differentially expressed in mice receiving urethane alone as compared to urethane plus immune stimulation. Up-regulated expression of TGFbeta3 and GM-CSF genes, in particular, was observed in leukocytes of urethane-exposed mice receiving immunostimulation. Interestingly, the cytokine products of these two genes were recently suggested as growth factors that may be related to reduction of fetal defects caused by teratogens. Genes for growth factors IGF-I, IGF-II and IL-2 were also identified as differentially expressed in urethane vs. urethane+immune stimulation mice, suggesting that these proteins should be considered for a potential contributing effect to reduced birth defects caused by immunostimulation.

Maternal immune stimulation in mice decreases fetal malformations caused by teratogens.

For unknown reasons, non-specific stimulation of the maternal immune system in pregnant mice has what appears to be a broad-spectrum efficacy for reducing birth defects. Immune stimulation by diverse procedures has proven effective, including footpad injection with Freund's complete adjuvant (FCA), intraperitoneal (IP) injection with inert particles to activate resident macrophages, IP injection with attenuated Bacillus Calmette-Guerin (BCG), and intrauterine injection with allogeneic or zenogeneic lymphocytes. Morphologic lesions that were significantly reduced included cleft palate and associated craniofacial defects, digit and limb defects, tail malformations, and neural tube defect (NTD). Teratogenic stimuli to induce these lesions included chemical agents (2,3,7,8-tetrachlorodibenzo-p-dioxin [TCDD], ethyl carbamate [urethane], methylnitrosourea [MNU], cyclophosphamide [CP], and valproic acid [VA]), physical agents (X-rays, hyperthermia), and streptozocin (STZ)-induced diabetes mellitus. Limited information is available regarding mechanisms by which such immune stimulation reduced fetal dysmorphogenesis. The collective literature suggests the possibility that immunoregulatory cytokines of maternal origin may be the effector molecules in this phenomenon.

[The study of some mechanisms of injurious effects of low-frequency noises]. / Issledovanie nekotorykh mekhanizmov povrezhdaiushchikh éffektov nizkochastotnykh shumov.

Low-frequency noise has been shown to cause certain functional changes in the organism of laboratory animals that manifest in changes of the state of the regulatory systems and metabolic disturbances at cellular and subcellular levels. The obtained data support the hypothesis of the mechanism of injurious effect of this physical factor including two basically correlated ways, i.e. the central mechanism associated with overexcitation of the hypotalamohypophysis-adrenal system and mediating the homeostatic parameters of the body, and the local one which is determined by a direct effect of low-frequency noise on highly organized structure of membraneous and genetic apparatus of cells.

In vitro methods for detecting cytotoxicity.

Toxicant-induced damage to cells (cytotoxicity) can depress cell growth, compromise intracellular metabolic processes, and/or cause loss of cell viability. Methods that indicate these cytotoxic changes following toxicant exposures are provided, including [³H]thymidine uptake to measure cell growth, MTT dye conversion to detect changes in metabolic activity, and trypan blue uptake to indicate loss of cell viability.

Nonspecific stimulation of the maternal immune system. I. Effects On teratogen-induced fetal malformations.

BACKGROUND: Maternal immune stimulation has reported, but unconfirmed, efficacy for reducing chemical-induced morphologic defects in mice. METHODS: Teratogenic chemicals (2,3,7, 8-tetrachlorodibenzo-p-dioxin [TCDD], ethyl carbamate [urethane], methylnitrosourea [MNU], or valproic acid [VA]) were given to pregnant mice to induce cleft palate (TCDD, urethane), digital defects (urethane, MNU), or exencephaly (VA). Before teratogen administration, the immune system of female mice was stimulated by intraperitoneal (IP) administration of pyran copolymer or attenuated bacillus Calmette Guérin (BCG), or by footpad injection with Freund's complete adjuvant (FCA). RESULTS: Fetal defects caused by all four chemicals studied were reduced by maternal immunostimulation, sometimes dramatically. In addition to reducing VA-induced exencephaly, immunostimulation with FCA resulted in fetal mice displaying anury (absence of tails). Activated maternal immune cells could not be detected in fetal circulation using flow cytometry and a fluorescent cell-tracking probe. CONCLUSIONS: For the chemicals tested, maternal immune stimulation has efficacy in reducing fetal defects. Immune protection against teratogenesis may be an indirect effect of maternal immune cell activation.

Nonspecific stimulation of the maternal immune system. II. Effects on gene expression in the fetus.

BACKGROUND: Maternal immune stimulation reduces malformations caused by chemical teratogens. Mechanisms for this effect are not known. Altered expression of regulatory molecules (e.g., transforming growth factor [TGF-beta], tumor necrosis factor-alpha [TNF-alpha]) has been reported in fetuses from immunostimulated mice, which may affect gene expression. Expression of selected genes that function to control proliferation, differentiation, or apoptosis was evaluated in chemical-exposed fetuses, with or without maternal immunostimulation. METHODS: Ethyl carbamate (urethane) was given to pregnant ICR mice on day 10 of gestation to induce cleft palate. Before teratogen administration, the immune system of the female mice was stimulated by footpad injection with Freund's complete adjuvant (FCA) or by intraperitoneal injection with interferon-gamma (IFN-gamma). RESULTS: Maternal immunostimulation with interferon-gamma (IFN-gamma) decreased severity of the cleft palate lesion caused by urethane, while FCA decreased both incidence and severity of cleft palate. Gestation day 14 fetuses from urethane-exposed mothers displayed decreased expression of cell cycle/apoptotic genes bcl2alpha, bcl2beta, pkCalpha, and p53 in fetal heads. Immune stimulation with IFN-gamma-normalized expression of bcl2alpha, bcl2beta, and pkCalpha to control levels. Urethane also decreased the ratio of expression of bclalpha/p53, bclbeta/p53, and pkCalpha/p53, while maternal injection with IFN-gamma restored these expression ratios to control levels. Maternal immunization with FCA also significantly increased bcl2alpha/p53, bcl2beta/p53, and pkCalpha/p53 gene expression ratios. CONCLUSIONS: These results suggest that (1) the maternal immune system may possess heretofore unrecognized regulatory activity in fetal development, and (2) protection against urethane-induced cleft palate may be mediated through maternal immune regulation of fetal gene expression.

Functional and ultrastructural cell pathology induced by fuel oil in cultured dolphin renal cells.

Investigations were undertaken to elucidate in a marine mammal renal cell culture system the toxicity and some of the mechanisms of cytopathology in a standardized preparation following exposure to No. 1 fuel oil. Cell survivability of a cultured SP1K renal cell line from the Atlantic spotted dolphin Stenella plagiodon was reduced in a dose-dependent manner after a 12-h exposure to fuel oil. Early morphologic changes reflecting cytotoxicity, as revealed by transmission electron microscopy, included enlarged rough endoplasmic reticula, cytoplasmic vacuolization, and degenerative cytoplasmic inclusions, but mitochondria remained resistant. Assessment of extracellular proton loss by microphysiometry of cultured cells revealed fuel oil-induced enhancement of proton loss that was dependent upon both protein kinase C and renal epithelial Na(+)/H(+) counter-transport functioning, as the specific inhibitors H-7 and amiloride reduced this stimulatory petroleum effect. Cell cycle progression and apoptosis (programmed cell death) were studied in dolphin renal cells exposed to fuel oil for 12, 24, and 48 hours. The toxicant increased the percentage of cells in GO/GI phase and decreased the percentage of cells in S phase starting after 24 hours. The number of cells undergoing early apoptosis was also increased after 24 hours.

Synthesis of turkey Pit-1 mRNA variants by alternative splicing and transcription initiation.

The gene encoding turkey Pit-1/GHF-1 (tPit-1) spans approximately 12 kilobases (kb) and consists of 7 exons. One exon, which is located between exons 2 and 3, is designated exon 2a and codes for 38 amino acids not found in mammalian Pit-1. Because all tPit-1 variants contain exon 2a, they are denoted with an asterisk (*) to distinguish them from comparable mammalian Pit-1s. Three tPit-1 variants are generated by alternative splicing and transcription initiation. Splicing of exon 1 to an alternative acceptor splice site in exon 2 results in a 28 amino acid insertion in tPit-1beta* relative to tPit-1*. A transcript unique to the turkey has been identified by RT-PCR and RNase mapping. This transcript, designated tPit-1W*, arises following transcription initiation upstream of the alternative acceptor splice site in exon 2. In turkey pituitary, the mRNA for the tPit-1* variant is the most abundant, the tPit-1W* variant is intermediate, and the tPit-1beta* variant is the least abundant.

Characterization of 10 KDA prolamin genes in Phyllostachys aurea (Bambusoideae, Poaceae).

Prolamin is the dominant class of seed storage protein in grasses (Poaceae). Information on the 10 kDa multigene family coding for prolamins characteristic of the bambusoid-oryzoid grasses is limited. Two genes encoding 10 kDa prolamin were cloned and sequenced in the bambusoid species Phyllostachys aurea to assess the sequence diversity of this gene family in the oryzoid-bambusoid grasses. The genes, ~417 bp in length, were 96% similar at the DNA sequence level, differing in 12 base substitutions dispersed throughout the sequence. Eight of these mutations were nonsynonymous, leading to amino acid substitutions in the coding region, and one was nonsense, producing an amber stop codon. One gene had an open reading frame (ORF) of 139 amino acids, while the other gene had a shorter ORF (106 amino acids) due to the presence of a stop codon in the coding region and, thus, represents a pseudogene. Deduced proteins showed amino acid composition similar to that of rice. The study underscores the overall conserved nature of this multigene family and reflects considerable sequence divergence at the DNA and amino acid levels between the Oryza and the Phyllostachys genes. The systematic implication of the data is discussed in light of the inconsistent placement of Oryza in the Bambusoideae or Oryzoideae.

[The pathogenesis of central nervous system functional disordersafter exposure to microwave radiation]. / Biokhimicheskie aspekty patogeneza narushenii funktsii tsentral'noi nervnoi sistemy pri mikrovolnovom obluchenii.

A rate of lipid peroxidation, content of biogenic amines and cyclic nucleotides were studied in various brain structures of 168 rats under conditions of microwave irradiation within 24 min at 46 mW/cm2. Total irradiation of animals was shown to result in distinct inhibition of monoaminergic activity of brain, especially of hypothalamus, in impairment of metabolic reactions, in exhaustion of the lipid antioxidative system of brain cortex and in stimulation of the contrainsular apparatus functions.

[The cyclic nucleotide content of the blood plasma in mice irradiated at different doses]. / Soderzhanie tsiklicheskikh nukleotidov v plazme krovi myshei, obluchennnykh v raznykh dozakh.

Cyclic nucleotide content of plasma was shown to increase after irradiation of mice within a wide range of doses. Compared was the dynamics of changes in the cyclic nucleotide content 24 hr following irradiation with supralethal doses.

[A preclinical study of the embryotoxic properties of cucumarioside and its effect on adult generative function]. / Doklinicheskoe issledovanie émbriotoksicheskikh svoistv kukumariozida i ego vliianiia na generativnuiu funktsiiu vzroslykh osobei.

The preclinical study of embryotoxicity of a new immunomodulator cucumariosid was carried out. Cucumariosid at long-term use exerts no effect on the generative function of rats, the general condition of pregnant females and the postnatal development of the offspring, possesses no teratogenic action. The use of the drug in the preimplantation period of pregnancy and the implantation period produces the contraceptive effect.

[The dynamic content of prostaglandins and cyclic nucleotides in the blood of irradiated dogs and monkeys]. / Dinamika soderzhanie prostaglandinov i tsiklicheskikh nukleotidov v krovi obluchennykh sobak i obez'ian.

In experiments with dogs and monkeys a study was made of the dynamics of the content of prostaglandins (PG) and cyclic nucleotides after gamma irradiation. In dogs irradiation with lethal doses (3.1 and 50 Gy) caused a short-term, evidently stress growth of cAMP, PGE and PGF2 alpha levels. At the height of radiation sickness PGF2 alpha and cGMP content decreased considerably. Irradiation of monkeys with a nonlethal dose of 3.2 Gy changed PGE and PGF2 alpha levels to a lesser extent, while concentrations of cyclic nucleotides varied but their ratio remained stable.

[The effect of an acoustic calling signal on the sex hormone level in the blood of white mice]. / Vliianie akusticheskogo prizyvnogo signala na uroven' polovykh gormonov v krovi belykh myshei.

The action was studied of synthesized acoustic signals, similar to the natural invocatory ones by their characteristics, on the level of sexual hormones in male and female mature laboratory mice, and also the dependence of these hormonal shifts on rhythmic organization of the artificial sound signals. As a result of the experiments it was convincingly shown that synthesized acoustic signal with the frequency of 3500 Hz (S-1) caused a functional load on the glands of internal secretion which was testified by statistically significant increase in the level of sexual hormones in the blood of males (by testosterone and extradiole) and female mice (by extradiole and progesterone). In order to clear up the role of rhythmic organization of signal studies were conducted with the application of synthetic acoustic signal (S-2), different from S-1. The analysis of the data revealed the absence of significant changes in the level of sexual hormones both in males and in females during S-2 sounding.