Epigenetic germline inheritance in mammals: looking to the past to understand the future.

Life experiences can induce epigenetic changes in mammalian germ cells, which can influence the developmental trajectory of the offspring and impact health and disease across generations. While this concept of epigenetic germline inheritance has long been met with skepticism, evidence in support of this route of information transfer is now overwhelming, and some key mechanisms underlying germline transmission of acquired information are emerging. This review focuses specifically on sperm RNAs as causal vectors of inheritance. We examine how they might become altered in the germline, and how different classes of sperm RNAs might interact with other epimodifications in germ cells or in the zygote. We integrate the latest findings with earlier pioneering work in this field, point out major questions and challenges, and suggest how new experiments could address them.

Transgenerational transmission and modification of pathological traits induced by prenatal immune activation.

Prenatal exposure to infectious or inflammatory insults is increasingly recognized to contribute to the etiology of psychiatric disorders with neurodevelopmental components, including schizophrenia, autism and bipolar disorder. It remains unknown, however, if such immune-mediated brain anomalies can be transmitted to subsequent generations. Using an established mouse model of prenatal immune activation by the viral mimetic poly(I:C), we show that reduced sociability and increased cued fear expression are similarly present in the first- and second-generation offspring of immune-challenged ancestors. We further demonstrate that sensorimotor gating impairments are confined to the direct descendants of infected mothers, whereas increased behavioral despair emerges as a novel phenotype in the second generation. These transgenerational effects are mediated via the paternal lineage and are stable until the third generation, demonstrating transgenerational non-genetic inheritance of pathological traits following in-utero immune activation. Next-generation sequencing further demonstrated unique and overlapping genome-wide transcriptional changes in first- and second-generation offspring of immune-challenged ancestors. These transcriptional effects mirror the transgenerational effects on behavior, showing that prenatal immune activation leads to a transgenerational transmission (presence of similar phenotypes across generations) and modification (presence of distinct phenotypes across generations) of pathological traits. Together, our study demonstrates for, we believe, the first time that prenatal immune activation can negatively affect brain and behavioral functions in multiple generations. These findings thus highlight a novel pathological aspect of this early-life adversity in shaping disease risk across generations.

Pathological brain plasticity and cognition in the offspring of males subjected to postnatal traumatic stress.

Traumatic stress in early-life increases the risk for cognitive and neuropsychiatric disorders later in life. Such early stress can also impact the progeny even if not directly exposed, likely through epigenetic mechanisms. Here, we report in mice that the offspring of males subjected to postnatal traumatic stress have decreased gene expression in molecular pathways necessary for neuronal signaling, and altered synaptic plasticity when adult. Long-term potentiation is abolished and long-term depression is enhanced in the hippocampus, and these defects are associated with impaired long-term memory in both the exposed fathers and their offspring. The brain-specific gamma isoform of protein kinase C (Prkcc) is one of the affected signaling components in the hippocampus. Its expression is reduced in the offspring, and DNA methylation at its promoter is altered both in the hippocampus of the offspring and the sperm of fathers. These results suggest that postnatal traumatic stress in males can affect brain plasticity and cognitive functions in the adult progeny, possibly through epigenetic alterations in the male germline.

Epigenetic regulation in neurodevelopment and neurodegenerative diseases.

From fertilization throughout development and until death, cellular programs in individual cells are dynamically regulated to fulfill multiple functions ranging from cell lineage specification to adaptation to internal and external stimuli. Such regulation is of major importance in brain cells, because the brain continues to develop long after birth and incorporates information from the environment across life. When compromised, these regulatory mechanisms can have detrimental consequences on neurodevelopment and lead to severe brain pathologies and neurodegenerative diseases in the adult individual. Elucidating these processes is essential to better understand their implication in disease etiology. Because they are strongly influenced by environmental factors, they have been postulated to depend on epigenetic mechanisms. This review describes recent studies that have identified epigenetic dysfunctions in the pathophysiology of several neurodevelopmental and neurodegenerative diseases. It discusses currently known pathways and molecular targets implicated in pathologies including imprinting disorders, Rett syndrome, and Alzheimer's, Parkinson's and Hungtinton's disease, and their relevance to these diseases.

The ability of oestradiol administration to regulate protein levels of oestrogen receptor alpha in the hippocampus and prefrontal cortex of middle-aged rats is altered following long-term ovarian hormone deprivation.

Beneficial effects of oestrogen administration on cognition are attenuated if treatment is initiated following long-term ovarian hormone deprivation. The mechanisms underlying this attenuation are unknown. The present study aimed to assess the effects of long-term ovarian hormone deprivation on the ability of subsequent oestradiol treatment to regulate oestrogen receptor (ER) alpha and ERbeta, and steroid receptor coactivator (SRC)-1 in the hippocampus and prefrontal cortex of middle-aged rats. In an initial experiment to assess oestradiol regulation of these proteins, 2-month-old rats were ovariectomised and immediately implanted with capsules containing cholesterol or oestradiol. Brains were collected 10 days later. In a second experiment, middle-aged (10-month-old) rats were ovariectomised or underwent sham surgeries. Five months later, sham-operated rats were ovariectomised and received oestradiol implants. Previously ovariectomised rats underwent sham surgeries and received oestradiol or cholesterol implants. Protein levels of ERalpha, ERbeta, and SRC-1 were measured following 10 days of oestradiol treatment using western blotting. In young animals, oestradiol treatment significantly increased ERalpha in the hippocampus and prefrontal cortex relative to control treatment. In middle-aged animals, immediate oestradiol treatment significantly increased ERalpha in hippocampus, but not the prefrontal cortex. However, delayed oestradiol treatment failed to significantly increase ERalpha protein levels in hippocampus, but did so in prefrontal cortex. Levels of ERbeta and SRC-1 were unaffected by oestradiol treatment in either brain area in either of the age groups. These data indicate that prolonged ovarian hormone deprivation alters the ability of subsequent oestradiol replacement to regulate ERalpha protein levels in brain areas important for cognition.

Long-term ovarian hormone deprivation alters the ability of subsequent oestradiol replacement to regulate choline acetyltransferase protein levels in the hippocampus and prefrontal cortex of middle-aged rats.

The role of oestrogen replacement therapy in preventing or delaying age-associated cognitive decline is controversial. Therapy success may critically depend on the time of treatment initiation following cessation of ovarian function. The present study aimed to assess, in middle-aged rats, whether the ability of oestradiol to modulate the cholinergic system depends on the timing of treatment initiation following ovariectomy. Using western blotting, protein levels of choline acetyltransferase (ChAT) were measured in the hippocampus and prefrontal cortex (PFC), which are both important areas with respect to cognitive function. In an initial experiment, we established the effects of oestradiol delivered via implanted capsules on ChAT levels in the hippocampus and PFC of young adult animals. In a second experiment, we tested the ability of the same oestradiol treatment paradigm to affect ChAT protein in 15-month-old middle-aged rats that had been ovariectomised either at the age of 10 months or at 15 months. In both experiments, rats were sacrificed 10 days after receiving implants and ChAT protein levels were measured. In both young adult and middle-aged animals, oestradiol treatment initiated immediately after ovariectomy significantly increased ChAT levels in the hippocampus but not in the PFC compared to cholesterol control treatment. However, when oestradiol treatment was initiated 5 months after ovariectomy, it failed to significantly increase ChAT levels in the hippocampus, but did so in the PFC. These data indicate that, after prolonged ovarian hormone deprivation, the ability of subsequent oestradiol treatment to modulate ChAT protein levels is altered in a site-specific manner.

Oxidation behavior of Ru/TiO2 and metallic Ru fine particles on heating in air.

Thermogravimetry (TG) and differential thermal analysis (DTA) were used to investigate the oxidation behavior of Ru/TiO2 and metallic Ru fine particles during heating in air in the range 20-1000 degrees C. Temperature ranges of the oxidation for two samples of Ru/TiO2 with the compositions (92 wt% Ru, 8 wt% TiO2) and (75 wt% Ru, 25 wt% TiO2) and for pure metallic Ru fine particle agglomerates were determined. It was assumed that after the partial oxidation of Ru in the sample containing 75 wt% Ru and 25 wt% TiO2 and in the pure metallic Ru a diffusion barrier was formed, preventing further oxidation of Ru in Ru/RuO2 and Ru/RuO2/TiO2 matrices. XRD and TEM were used for the sample characterization.

The interaction of Prussian blue and dissolved hexacyanoferrate ions with goethite (alpha-FeOOH) studied to assess the chemical stability and physical mobility of Prussian blue in soils.

Colloidal Prussian blue and dissolved hexacyanoferrate ions strongly interact with the surface of goethite (alpha-FeOOH) particles. Whereas Prussian blue is deposited on the surface of goethite as a solid phase with all the properties typical of solid Prussian blue, the hexacyanoferrate ions form initially a layer of surface bound hexacyanoferrate ions, which exhibit properties intermediate between those of free hexacyanoferrate ions and hexacyanoferrate ions bound in Prussian blue. The products of interaction of goethite with Prussian blue and hexacyanoferrate ions were studied by voltammetry of immobilized microcrystals, infrared spectrometry, Vis diffuse reflection spectrometry, transmission electron microscopy, and ESR spectroscopy. The kinetics of Prussian blue destruction was measured by solution Vis spectrometry and also by voltammetry of solid microcrystals. The remarkable stability of Prussian blue in soils can be explained by its strong adherence as a solid phase to iron oxides and oxide hydroxides in soils. This prevents the formation of soluble colloids of Prussian blue. It does not prevent the hydrolysis of Prussian blue to iron oxide hydroxides and hexacyanoferrate(II) ions at elevated pH which proceeds at almost the same rate as that of pure Prussian blue colloid. Even when this hydrolysis of Prussian blue occurs, it is the hexacyanoferrate(II) that is effectively adsorbed on the surface of iron oxide hydroxides, again decreasing the physical mobility of hexacyanoferrate.

Enhancement by carboxymethylglucan of early cellular damage in 1 Gy-irradiated mice.

Our results describe a novel carboxymethylglucan (CMG) activity, namely its radiosensitizing effect on early cellular damage in mice gamma-irradiated with a dose of 1 Gy. An increase of thymidine levels in blood plasma, determined 4 h after irradiation, was used as an indicator of the early cell death. The radiosensitizing effect was observed when administering CMG at time intervals close to irradiation time (1 h before to 1 h after irradiation). Diclofenac (an inhibitor of prostaglandin production) had no modifying effects on elevation of plasma thymidine levels induced by radiation or radiation + CMG. Pentoxifylline (an inhibitor of synthesis of tumour necrosis factor and of phosphodiesterase) administration elevated plasma thymidine to similar levels as CMG alone, combined pentoxifylline + CMG treatment had not additive effects.

Postirradiation administration of adenosine monophosphate combined with dipyridamole reduces early cellular damage in mice.

The administration of dipyridamole and adenosine 5'-monophosphate (AMP) to mice 5 to 25 min after 1 Gy of total-body gamma irradiation was found to decrease cellular damage, as indicated by the thymidine level in plasma and the amount of saline soluble polynucleotides in the thymus. The drug combination used did not influence similar cytotoxic effects of hydrocortisone. Furthermore, it was shown that the addition of dipyridamole and AMP to in vitro irradiated suspensions of thymocytes enhanced the rejoining processes of DNA strand breaks. Receptor-mediated action of extracellular adenosine may be responsible for the therapeutic effects observed.

Protection of early cellular damage in 1 Gy-irradiated mice by the elevation of extracellular adenosine.

In whole-body 1Gy-irradiated mice a modification of early cellular damage by means of preirradiation dipyridamole and adenosine monophosphate (AMP) treatment was investigated. Both drugs were given either alone or in combination, AMP being administered i.p. at doses of 5, 10 and 15 mg, dipyridamole s.c. at the dose of 2 mg, 20 min before AMP. The thymidine level in plasma and the amount of free polynucleotides in the thymus and spleen, both estimated at the interval of 4 h after irradiation, were used as indices of early cellular damage in vivo. The elevated level of thymidine observed in the plasma of irradiated controls decreased significantly after the administration of AMP (5 mg) alone to 71%, after the combination of dipyridamole and AMP a still deeper significant fall to 60% was observed. Such a protective effect was observed when injecting AMP 15 min before irradiation. Using the interval of 65 min between AMP administration and irradiation, no protection was detected. The higher doses of AMP (10, 15 mg) enhanced the protective effect manifested in plasma thymidine level only moderately. The amount of free polynucleotides, elevated in the thymus and spleen of irradiated mice, was significantly decreased in the thymus of mice pretreated with the combination of dipyridamole and AMP. The results suggest that the treatment used decreases the radiation damage of the sensitive thymocyte population. It is proposed that the joint use of AMP, an adenosine prodrug, and dipyridamole, a drug inhibiting adenosine uptake by cells, leads to an elevation in extracellular adenosine which activates cell surface adenosine receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Long-term sealing ability of a calcium hydroxide sealer.

A calcium hydroxide sealer (Sealapex) was compared with a zinc oxide and eugenol sealer (Tubli-Seal) over a 32-wk interval to examine solubility in an in vitro simulation. After obturation with gutta-percha and the appropriate sealer, specimens were immediately immersed in a saline solution to challenge the sealers' solubility. The solutions were changed weekly to allow for a continued dissolution of the sealers and to prevent establishment of an equilibrium between the solution and the sealers. The 2- and 32-wk specimens were removed from the solutions, immersed in India ink for 3 days, and then made transparent by a clearing process. Microscopic examination was used to determine the linear penetration of the ink for each tooth. Results revealed that Sealapex statistically had no greater dissolution (based upon linear penetration) than Tubli-Seal at both 2 and 32 wk. It is suggested that Sealapex has a sealing ability comparable to Tubli-Seal and can withstand long-term exposure to tissue fluids without significant leakage. This may allow time for the biochemical action of the calcium hydroxide to stimulate physiological calcification of the apical foramen.

The effect of cyclophosphamide and gamma irradiation on adenosine deaminase and purine nucleoside phosphorylase in mice.

Changes in ADA and PNP activities in the spleens and thymuses of mice were studied after a single administration of cyclophosphamide (CY, 200 mg/kg) and after whole-body gamma irradiation (5.5 Gy), applied alone or three days after CY application. In the first days after the treatment the enzyme activities were significantly depressed (p less than 0.01) with the exception of ADA in the spleen, where a high elevation (220-380%) in relation to controls was observed. During the regeneration period a pronounced rise of PNP activity in the spleen occurred mainly after a combined application of CY and irradiation (270%). In the thymus the regeneration was manifested by a mild increase of both ADA and PNP activities towards control values. The findings suggest that the expressive changes of ADA and PNP activities, participating in the purine salvage pathway, may, after a cytotoxic treatment, influence the nucleotide pool and DNA synthesis in lymphoid organs.

Purine metabolizing enzyme activities in radiosensitive tissues of mice after sublethal whole-body irradiation.

The activities of adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) were determined between days 1-14 in the spleen, thymus and femoral bone marrow of mice subjected to whole-body gama irradiation with a dose of 5.5 Gy. In control animals, the highest activity of ADA (as related to 10(6) cells) was recorded in the thymus (58.9 pmol.s-1), the lowest one in the femur (34.8 pmol.s-1), the PNP activity was the lowest in the thymus (14.5 pmol.s-1) and the highest in the femur (96.0 pmol.s-1). In the spleen, an elevation of ADA activity (up to 379%) was observed during the first postirradiation days; PNP activity was reduced (to 58%) on postirradiation day 3, followed by the return and even elevation on day 14 (265%). In the thymus, a parallel reduction of the activities of both enzymes appeared during the first postirradiation days, with a subsequent increase during the regeneration phase. In the femoral bone marrow, ADA and PNP activities were increased on postirradiation day 1 (275% and 201%, respectively). Reference is made to the possible relationship between the observed characteristic changes in activities and the degree of damage and/or renewal of cell population in the hemopoietic tissues after irradiation.

Determination of adenosine deaminase and purine nucleoside phosphorylase activities using high-performance liquid chromatographic and radiochromatographic methods.

Two methods for the determination of adenosine deaminase and purine nucleoside phosphorylase activities were compared. The high-performance liquid chromatographic (HPLC) technique used separation on a reversed-phase silica column and exhibited adequate sensitivity and a markedly higher rate of analysis compared with that of the paper radiochromatographic method. Correlation analysis of the results obtained by the two methods on a set of lymphoid cells from 25 patients with lympho-proliferative disorders confirmed the utility of the HPLC technique in clinical investigations.

The effect of hypoxia on the activity of purine nucleoside phosphorylase in rats.

The activity of purine nucleoside phosphorylase (PNP) in rat erythrocytes fractionated by centrifugation in microhematocrit capillaries was studied. After seven-day hypoxia (54 kPa) the PNP activity was increased by 67 +/- 4% (S.E.M.) in the lightest fraction of erythrocytes; on the fifth day after hypoxia PNP activity did not differ from control values.

The preparation of 59Fe-labelled transferrin of high radioactivity for ferrokinetic studies on small laboratory animals.

A modified method of Cavill for the separation of the complex of isotopically labelled iron and transferrin from unbound iron residues is described. The method employing an ion exchanger permits to obtain preparations with a high radioactivity per volume unit of solution, which is necessary for carrying out ferrokinetic studies on small laboratory animals.

Actinobacilli in domestic fowl.

Reports on haemolytic rods from salpingitis in hens and from organs of died chicks may be found since the year 1950 (Kjos-Hanssen). Correct systematic classification on these microorganisms is still lacked because not all relations to the nearest pasteurellae were known as well as the clear differences in the Pasteurella-Actinobacillus group. The authors have isolated 34 suspected strains from 14 localities; 25 strains were from salpingitis or internal organs of died fowl inclusively chicks, and 9 strains from choanas of pullets clinically healthy. Bacteriological, serological, chemical and biological tests gave the results as follows: 1) All strains exhibit fundamental properties of Pasteurella-Actinobacillus group, and moreover, they show ability to grow on MacConkey agar with crystal violet (BioQuest) being a significant feature of the genus Actinobaccillus (Mráz, 1975). 2) From the nearest species Actinobacillus haemolyticus (Newsom and Cross, 1932) Mráz, 1969, they differ with expressive haemolysis on agar with sheep blood, single haemolytic zone on agar with lamb blood according to Smith (1962), structure of somatic antigen, with natural hosts (gallinaceous birds) and pathogenicity for 5-day-old chicks. The GC content in DNA was determined in the range 39,6-42,9% (mean value 41,5%). 3) The first who presumed an independent state of these microorganisms, was Kohlert (1968), from the work of which the epithet for correct name, i.e. Actinobacillus salpingitidis (Kohlert, 1968) comb. nov., was accepted.