Role of Oestrogen α Receptors in Sociosexual Behaviour in Female Rats Housed in a Seminatural Environment.

The present study investigated the role of oestrogen receptor (ER)α in the ventromedial nucleus of the hypothalamus (VMN), the preoptic area (POA), the medial amygdala (MePD) and the bed nucleus of stria terminalis (BNST) in sociosexual behaviour in female rats. This was conducted in two sets of experiments, with the VMN and POA investigated in the first set, and the MePD and BNST in the second set. The VMN and POA received intense projections from the MePD and BNST. We used a short hairpin RNA encoded within an adeno-associated viral vector directed against the gene for ERα to reduce the number of ERα in the VMN or POA (first set of experiments) or in the BNST or MePD (second set of experiments) in female rats. The rats were housed in groups of four ovariectomised females and three males in a seminatural environment for 8 days. Compared with traditional test set-ups, the seminatural environment provides an arena in which the rats can express their full behavioural repertoire, which allowed us to investigate multiple aspects of social and sexual behaviour in groups of rats. Behavioural observation was performed after oestrogen and progesterone injections. A reduction of ERα expression in the VMN or POA diminished the display of paracopulatory behaviours and lordosis responses compared to controls, whereas the lordosis quotient remained unaffected. This suggests that ERα in the VMN and POA play an important role in intrinsic sexual motivation. The reduction in ERα did not affect the social behaviour of the females, although the males sniffed and pursued the females with reduced ERα less than the controls. This suggests that the ERα in the VMN and POA is involved in the regulation of sexual attractiveness of females. The ERα in the MePD and BNST, on the other hand, plays no role in sociosexual behaviour.

Induction of circular episomes during rescue and replication of adeno-associated virus in experimental models of virus latency.

The synthesis of linear duplex replicative structures (monomers, head-to-head, and tail-to-tail dimers) is an important hallmark of the productive phase of the adeno-associated virus (AAV) life cycle. These structures are generated by a strand-displacement replication mechanism and believed to be a reservoir for single-stranded DNA genomes. During the course of studies with recombinant versions of AAV (rAAV), we discovered the assembly of circular duplex provirus derivatives in latently infected cell lines under conditions permissive for replication (i.e., helper virus dependent). These novel structures were cloned by bacterial trapping revealing a markedly homogeneous structure that included a single copy of the rAAV genome joined head-to-tail about the inverted terminal repeats (ITR). Restriction and sequence analysis of the point of circularization revealed a so-called "TRT" domain, consisting of a single ITR hairpin palindrome flanked by 5' and 3' D sequence elements. The circular conformation was additionally characterized by Southern blotting and confirmed by purification on an ethidium bromide-CsCl gradient where the buoyant density was consistent with circular supercoiled DNA. These findings suggest that AAV replication is accompanied by the assembly of circular duplex structures.

Inhibitory effect of melatonin on 7, 12-dimethylbenz[a]anthracene-induced carcinogenesis of the uterine cervix and vagina in mice and mutagenesis in vitro.

Forty female CBA mice aged 3-4 months were exposed twice a week during 2 months to intravaginal applications of polyurethane sponges impregnated with 0.1% solution of 7,12-dimethylbenz[a]anthracene (DMBA) in triethyleneglycol. Three hours after each application the sponges were taken out. Starting from the day of the 1st DMBA application a part of mice was exposed five times a week during 4 months with melatonin in tap water (20 mg/l) given at night time (from 18:00 to 09:00 h). Additional 20 female CBA mice were intact and served as a control. All mice were sacrificed in 6 months after start of the experiment. Seven of 20 mice exposed to DMBA alone developed malignancies in the vagina and cervix uteri and two mice developed benign cervical tumors. No malignancies in vagina and uterine cervix and three vaginal papillomas were observed in mice exposed to DMBA+melatonin. There were no any tumors in intact controls. Two in vitro tests were used for mutagenicity studies: the Ames test (strains TA 97 and TA 98 of Salmonella typhimurium) and the single cell gel electrophoresis assay (SCGE assay or COMET assay) performed on CHOK1 cells. In tested strains melatonin significantly reduced the mutagenicity of DMBA. In the SCGE assay preincubation with melatonin led to a strong inhibition of clastogenic activities of DMBA. Thus, our data indicate that pineal indole hormone melatonin inhibits cervical and vaginal carcinogenesis induced by DMBA in mice and possess antimutagenic and anticlastogenic effect in vitro.

Effects of melatonin on N-nitroso-N-methylurea-induced carcinogenesis in rats and mutagenesis in vitro (Ames test and COMET assay).

The effect of melatonin, an indole hormone of the pineal gland, on the initiation of N-nitroso-N-methylurea (NMU)-induced carcinogenesis in rats and mutagenesis in vitro has been investigated. Two-month-old female LIO rats (groups 1 and 2) were exposed to a single injection of NMU (50 mg/kg of body weight, i.v.). Rats from group 2 were given melatonin orally (20 mg/l) from 18:00 to 09:00 h over 3 days (2 days before and 1 day after NMU injection). Animals from group 1 (control) were administered the solvent (ethanol/water, 1:1000). Rats were followed up to natural death or were sacrificed when moribund. Tumors developed both in rats treated with NMU alone (50.0%) and in rats exposed to NMU plus melatonin (34.8%). The percentage of malignant tumor-bearing rats in group 2 (21.7%) was lower (P < 0.02) than that in the other group (41.7%). Melatonin also decreased the multiplicity of malignant tumors 1.3-fold and reduced the incidence of malignancies in some organs. Two in vitro tests were used for mutagenesis studies: the Ames test (strains TA 100 and TA 102 of Salmonella typhimurium) and the Single Cell Gel Electrophoresis assay (SCGE assay or COMET assay) performed on CHOK1 cells. Melatonin itself revealed no genotoxic effect in either of the tests. No protective action of melatonin (at doses of up to 2 micromol/plate) towards NMU was found in the Ames test. In contrast, in the SCGE assay a slight, but statistically significant (P < 0.001), dose-related anticlastogenic effect of melatonin (10(-10)-10(-7) M) was observed. Thus, our data indicate that melatonin may act as an anti-initiating hormone in NMU-induced carcinogenesis and possess anticlastogenic activity towards NMU in CHOK1 cells.

Modulatory effects of melatonin on genotoxic response of reference mutagens in the Ames test and the comet assay.

The effect of a potent endogenous antioxidant, the pineal gland indole melatonin (MLT) on the mutagenicity of twelve well-known mutagens and carcinogens has been investigated using two in vitro tests the Ames test and the single cell gel electrophoresis assay (SCGE assay or COMET assay). The 12 mutagens used were 7, 12-dimethylbenz(a)anthracene (DMBA), benzo(a)pyrene (BP), 2-aminofluorene (AF), 1,2-dimethylhydrazine (DMH), bleomycin, cyclophosphamide (CP), 4-nitroquinoline-N-oxide (NQO), 2,4, 7-trinitro-9-fluorenone (TNF), 9-aminoacridine (AA), N-nitrosomethylurea (NMU), mitomycin C and sodium azide tested in the absence or in the presence of S9 mix. MLT alone turned out neither toxic nor mutagenic in the Ames test and revealed clastogenic activity at the highest concentration tested (100 microM) in the SCGE assay. In four Salmonella typhimurium tester strains TA 97, TA 98, TA 100 and TA 102 MLT significantly reduced the mutagenicity of chemicals which require S9 activation. In the SCGE assay performed on CHO cells, preincubation with MLT led to a strong inhibition of clastogenic activities of DMBA and CP, and in a lesser extent with BP and NMU. With mitomycin C, MLT exacerbated responses in both tests. The possible mechanisms of MLT's inhibitory action are discussed.

[The influence of melatonin on mutagenicity and antitumor action of cytostatic drugs in mice]. / Vliianie melatonina na mutagennost' i protivoopukholevyi éffekt tsitostatikov u myshei.

SHR mice received single injections of N-nitrosomethylurea (NMU, 50 mg/kg, i.p.), cyclophosphamide (CP, 200 mg/kg, i.p.) or 1,2-dimethylhydrazine (DMH, 15 mg/kg, s.c.) alone or in combination with melatonin (5 mg/kg, s.c.). For mutagenic study chromosome aberrations tests (ChA) in bone marrow cells and sperm head anomaly test (SHA) were used. Melatonin did not appear mutagenic in either of the tests and significantly reduced the level of ChA (%) from 16.9 +/- 1.6 (NMU), 13.7 +/- 3.5 (CP) and 8.7 +/- 0.3 (DMH) to 4.5 +/- 0.6, 4.3 +/- 0.9 and 5.6 +/- 0.2, respectively, (p < 0.05). Similarly, SHA frequency (%) under the melatonin influence was reduced from 18.6 +/- 0.4 (NMU), 17.7 +/- 0.4 (CP) and 10.0 +/- 0.5 (DVH) to 9.9 +/- 0.5, 6.1 +/- 0.3 and 7.5 +/- 0.2, respectively, (p < 0.05). Unlike in controls, exposure to melatonin in drinking water (20 mg/l, at night) or in injections (5 mg/kg, s.c.) alone or in combination with NMU or CP failed to influence subcutaneously-transplanted Ehrlich carcinoma growth. These findings suggest that melatonin reduced the mutagenicity of the cytostatic drugs without affecting their anti-tumor action.

[Geographic variability in the sexually mature phase of Ixodes persulcatus (Ixodidae). Experience in using databases on morphometry]. / Geograficheskaia izmenchivost' polovozreloi fazy Ixodes persulcatus (Ixodidae). Opyt primeneniia baz dannykh po morfometrii.

The aim of the article is to estimate geographic variation of Ixodes persulcatus adults as a whole. Intraspecific variation of the females and males of Ixodes persulcatus Schulze, 1930 has been studied in eight geographical localities ("populations") of its distribution range (fig. 1.). The distance between western and eastern localities is more than 8700 km, between northern and southern ones it is approximately 2900 km. Twenty five to thirty specimens of each sex were studied in each geographical locality. The following eleven characters were used (fig. 2, 3): length of scutum (conscutum), width of scutum (conscutum), length of anal ring, width of anal ring, length of spiracular plate, width of spiracular plate, length of gnathosoma, width of gnathosoma, length of II-III articles of palps, length of hypostome, length of tarsus 1. The multidimensional scaling method by means of software package SYSTAT was used for estimation of relationships between populations on the basis of morphometrical data. The differences between the populations were revealed only from absolute sizes of organs, whereas their proportions (i. e. shape) were constant in all geographical localities. Fig. 4, 1 shows that females from localities G (Primorski Territory) and C (Tien Shan Mountains) occupy extreme positions. Fig. 4, 2 shows that males from localities G (Primorski Territory) and D (SW Altai Mountains) on the one hand and A, B (European) on the other one occupy extreme positions. Locality C (Tien Shan Mountains) is similar to F (Western Sayan Mountains) and to European (A, B) whereas females of locality C differ from A, B and F. Taking into account the partial discrepancy of relationships between populations in sexes we have united the data on corresponding characters of both sexes in the aggregate data base (fig. 4, 3). This was possible owing to the multidimensional scaling method. Fig. 4, 3 shows isolated position of the population G (Primorski Territory), specimens of which are the largest in sizes. The populations D (SW Altai Mountains) and H (Sakhalin Island) are morphometrically most similar to the population G. The population C (Tien-Shan Mountains) is represented of the smallest specimens. The European populations (A and B) are closer to C. The largest sizes are typical of the populations G and D associated with relict Tertiary landscapes of Primorski Territory and SW Altai with which areas of ecological optimum of I. persulcatus coincide. The smallest sizes are observed in the European populations (A, B) near the north-western boundary of the distribution range of the species, as well as in the Alpine population of Tien-Shan Mountains, near the upper vertical boundary of the distribution range (2000-3000 m above sea level). Climatic conditions of the habitat in these areas are similar to those of the northwestern part of the distribution range of I. persulcatus.