Insecticidal and binding activities of N3-substituted imidacloprid derivatives against the housefly Musca domestica and the alpha-bungarotoxin binding sites of nicotinic acetylcholine receptors.

N3-substituted imidacloprid congeners containing C1-C6 alkyl groups or various analogous groups, and their corresponding nitromethylene analogues, were used in this study. Their insecticidal activity against the housefly, Musca domestica, and their binding activity toward the nicotinic acetylcholine receptor were determined. The insecticidal test was conducted using the synergists piperonyl butoxide and propargyl propyl phenylphosphonate. The binding assay was performed with housefly head membrane preparations using radio-labelled alpha-bungarotoxin. Both insecticidal and binding activities were drastically lowered by the introduction of alkyl/allyl groups at the imidazolidine NH sites of both nitroimino and nitromethylene compounds. The binding activity of N3-substituted nitromethylene analogues was much higher than that of the corresponding nitroimino analogues. However, the insecticidal activity of both series of compounds with a given substituent was nearly identical. The insecticidal activity correlated positively with the binding activity after taking into account the structural difference of the nitroimino and nitromethylene moieties and a structural feature of the N3-substituents.

Transplantation of cryopreserved mouse, Chinese hamster, rabbit, Japanese monkey and rat ovaries into rat recipients.

Partial ovaries from mice, hamsters, rabbits, Japanese monkeys and rats have survived deep-freezing and returned to a normal morphological state after being thawed and transplanted into the rat uterine cavity. This report describes the ice-free cryopreservation of mouse and other ovaries at -196 degrees C by vitrification. The vitrification solution was based on the solutions reported by Rall & Fahy [16]. After ovaries had been exposed to the vitrification solution, they were frozen, with their suspending medium, by liquid nitrogen. After freezing, the ovaries were thawed in 37 degrees C water. The viability of the previously frozen ovarian tissue was tested by transplanting it into the uterine cavity of pseudopregnant rats. Seven days after transplantation, the ovaries were removed with the rat uterus, and stained with haematoxylin and eosin for histological examination. Survival of the frozen-thawed the ovaries in the rat uterine cavity demonstrates that these ovaries can tolerate exposure to osmotic dehydration and vitrification in a concentrated solution of cryoprotectant and are then immunologically acceptable to the uterine cavity.

5-Azidoimidacloprid and an acyclic analogue as candidate photoaffinity probes for mammalian and insect nicotinic acetylcholine receptors.

The 5-azido analogue of the major insecticide imidacloprid, 1-(5-azido-6-chloropyridin-3-ylmethyl)-2-nitroiminoimidaz olidine (1), and an acyclic analogue, N-(5-azido-6-chloropyridin-3-ylmethyl)-N'-methyl-N' '-nitroguanidine (2), were prepared in good yields as candidate photoaffinity probes for mammalian and insect nicotinic acetylcholine receptors (nAChRs). The essential intermediate was 5-azido-6-chloropyridin-3-ylmethyl chloride (3) prepared in two ways: from 6-chloro-5-nitronicotinic acid by selective reduction and then diazotization, and from N-(6-chloropyridin-3-ylmethyl)morpholine by an electrophilic azide introduction with lithium diisopropylamide followed by chlorine substitution of morpholine with ethyl chloroformate. Coupling of 3 with 2-nitroiminoimidazolidine gave 1. Conversion of 3 to 2 was achieved in good yields via the hexahydrotriazine intermediate 14. Fortuitously, the azido substituent in 1 and 2 increases the affinity 7-79-fold for rat brain and recombinant alpha4beta2 nAChRs (K(i)s 4.4-60 nM competing with [(3)H](-)-nicotine) while maintaining high potency on both insect nAChRs (Drosophila and Myzus) (K(i)s 1-15 nM competing with [(3)H]imidacloprid). Azidopyridinyl compounds 1 and 2 are therefore candidate photoaffinity probes for characterization of both mammalian and insect receptors.

Inhibitory effects of nitric oxide on the expression and activity of aromatase in human granulosa cells.

The aim of the present study was to explore the mechanisms by which nitric oxide (NO) may inhibit aromatase activity of human granulosa cells. Ovarian granulosa-luteal cells, obtained from patients undergoing in-vitro fertilization (IVF) were cultured in the presence of NO-related substances. After 24 h of culture, aromatase activity of the cells was significantly inhibited by treatment with the NO donors, SNAP or NOC12 at > or =10(-4) M in a dose-dependent manner. Treatment with NO catabolites or a peroxynitrite-releasing compound, SIN1, had no significant influence. Treatment with SNAP at 10(-3) M decreased relative aromatase mRNA values by 72% (P<0.05) and intracellular cyclic AMP concentrations by 53% (P<0.01). However, treatment with H89, an inhibitor of protein kinase A, did not inhibit aromatase activity. Since there were no significant effects of NO catabolites or peroxinitrite, the inhibitory action of NO donors on aromatase must be related to NO release. The action of NO is, in part, attributable to the down-regulation of aromatase gene transcription. Although NO decreased intracellular cAMP values, down-regulation of aromatase gene transcription may not be mediated by protein kinase A-dependent mechanisms.

Evaluation and characterization of congenital hypothyroidism in rdw dwarf rats.

The rdw rat is a new strain of dwarf mutant that has decreased blood thyroxine (T4) and growth hormone (GH) concentrations and testicular enlargement during development and aging. To confirm whether this strain can be used as a new hypothyroid model, the experiments reported here were carried out, using adult rdw rats, rdw rats treated with thyroxine, and clinically normal (N) Wistar-Imamichi rats. Clinical parameters of deficient thyroid function in rdw rats were chosen for evaluation and characterization. Body weight, hemoglobin (Hb) concentration, hematocrit (Hct), glucose (GLU), and systolic blood pressure were significantly lower, and serum values for aspartate transaminase (AST), total cholesterol (TC), total protein (TP), and blood urea nitrogen (BUN) were higher in rdw than in N rats. Serum concentrations of total T4 and free triiodothyronine (FT3) were significantly lower, and serum thyroid-stimulation hormone (TSH) concentration was markedly higher in rdw than in N rats. Serum GH concentration was significantly lower in rdw than in N rats. Results of histologic examination indicated that the thyroid gland of rdw rats was markedly atrophied, compared with that of N rats. Results of clinical examination of organs and hematologic and biochemical values in rdw rats corresponded to those of the hypothyroid state in humans. Most organ weights (heart, kidney, spleen, and adrenal gland), hematologic and biochemical values (Hb, Hct TC, TP, BUN), blood pressure, and serum hormone (TSH and GH) values underwent substantial restoration (partial or complete) toward normal in response to replacement therapy. In conclusion, the rdw rat is a useful model of congenital hypothyroidism.

Imidacloprid and Related Compounds: Structure and Water Solubility of N-Alkyl Derivatives of Imidacloprid.

An intramolecular hydrogen bond between NH•••O2N in insecticide, imidacloprid (1), and its nitromethylene analog 15 was proved by NMR and IR spectra. That electron delocalization over their planar moieties was disrupted by alkylation at the imidazolidine nitrogen atom is demonstrated by the hypsochromic shifts in UV and deshielding effect in NMR spectra. Interestingly, the N-alkyl derivatives (C1-5) had greater water solubility than 1, although increasing alkyl chain length decreased the solubility. The hydrophilicity of the alkyl derivatives would result from remote charge heads being formed as a result of the conjugation disruption by alkylation, while the hydrophobicity of 1 could be ascribed to the charge distribution over the conjugated system coupled with the intramolecular H-bonding. The greater water solubility of 15 than 1 and contrastively small solubility of the cyanoimine analogue are discussed based on the difference in their steric crowding.

Possible contribution of follicular interleukin-1beta to nitric oxide generation in human pre-ovulatory follicles.

The aim of this study was to investigate the relationships between follicular nitric oxide (NO) metabolite concentrations and several related variables, with special reference to follicular interleukin-1beta (IL-1beta). The follicular fluid from the leading and secondary follicles was collected individually from 20 women undergoing in-vitro fertilization (IVF) treatment, and the concentrations of nitrite (NO2-) and nitrate (NO3-) were determined fluorometrically using 2,3-diaminonaphthalene. Both follicular nitrite (r = 0.42, P < 0.01) and nitrate (r = 0.49, P < 0.001) were found to be significantly correlated with follicular IL-1beta concentrations. There were also significant positive correlations between follicular nitrate and the number of oocytes retrieved (P < 0.01) and serum oestradiol concentration on the day of human chorionic gonadotrophin (HCG) administration (P < 0.05). When follicular cells were incubated in vitro with 10 ng/ml of IL-1beta for 24 h, nitrate generation was significantly (P < 0.01) elevated compared with the control. In conclusion, our study demonstrates that follicular IL-1beta and the number of developing follicles are significant variables that affect follicular NO concentrations, and points to the possible contribution of IL-1beta to NO generation in human preovulatory follicles.

No effect of voluntary exercise on ovarian follicle in rats.

The present study was to clarify the relationship between voluntary exercise and follicular growth or ovulation. Rats kept for 4 weeks in a rotating drum with free access to the wheel, food and water ran 2-12 km per day. The number of ova shed after superovulation treatments and the number of non-atretic follicles were not influenced by voluntary exercise. These experiments demonstrate that spontaneous voluntary exercise does not affect either the number of ova shed or the number of non-atretic follicles in superovulating rats or control rats.

Increase in testis weight of hereditary dwarf rats (rdw/rdw) with advancing age.

Testis weight and seminal vesicle weight (relative to total body weight) in rdw rats and N rats were compared with advancing age. The results obtained were as follows 1) The increase in testis weight became remarkable at 15-16 weeks of age. The weight remained constant up to 23-30 weeks of age and decreased thereafter with age. 2) Seminal vesicle weight increased rapidly in rdw rat at 15-16 weeks of age, reflecting elevated androgen secretion and sexual maturation. 3) From the viewpoint of histology, one of causes of the increase in testis weight may be edema. The increase in testis weight coincided with the increase in androgen secretion at 15-16 weeks of age as suggested by the relative increase in seminal vesicle weight.

Intrauterine transplantation of isogenic pancreatic islets in experimental diabetic rats.

The effect of intrauterine transplantation (IU group) as a potential immunologically privileged site on the diabetic state of the recipient was compared with that of conventional intraperitoneal transplantation (IP group) using Fisher 344 rats. Islets were isolated from the pancreata of normal rats and transplanted into the uterus and peritoneal cavity of the isogenic rats with experimental diabetes, which were treated with estradiol benzoate and progesterone. Although all the rats in both groups became normoglycemic within 4 days after transplantation, all of those in the IU group relapsed into a diabetic state up to the 20th day after transplantation. On the other hand, 6 of 8 rats in the IP group remained normoglycemic throughout the experimental period. Weight gain and diminution of urinary glucose excretion in the IU group were significantly lower than those in the IP group (P < 0.01). The glycosylated hemoglobin level in the IU group did not differ significantly from that in the IP group, but the serum level of fructosamine in the IU group was significantly higher than that in the IP group (P < 0.01). These results indicate that the response to fluctuations of blood glucose of islets in the uterine cavity is less than that of islets in the peritoneal cavity. Histologically, islets were observed to be aggregated in the uterine cavity, however the number of cells decreased markedly with time. Although this study demonstrated that blood glucose was normalized by transplantation of islets into the uterine cavity of diabetic rats, long-term survival of the islets in this location was not obtained.

Pituitary and serum levels of prolactin (PRL), thyroid stimulating hormone (TSH) and serum thyroxine (T4) in hereditary dwarf rats (rdw/rdw).

Female and male hereditary dwarf mutation rats (rdw), offspring of those found by Koto et al. in a breeding colony of Wistar-Imamichi rats, were decapitated to collect blood and pituitary glands at 10 weeks of age. Levels of pituitary prolactin (PRL), thyroid stimulating hormone (TSH) and serum PRL, TSH and thyroxine (T4) in rdw rats were measured and compared with levels in normal rats (N). The hormone levels were measured with radioimmunoassay (RIA). It was found that the levels of pituitary and serum PRL were considerably lower in rdw rats and that the level of serum T4 was also significantly lower than that in normal rats. TSH per pituitary was significantly lower in rdw rats but TSH per mg pituitary was not different between rdw and normal rats. The levels of serum TSH in rdw was not different from that in normal rats, but was rather higher in female rdw rats than in normal female rats. When the results of previous research were combined with the present results, it was clear that rdw rats are characterized by hypoplasia of GH and PRL synthetic cells of the pituitary and that rdw rat is possibly useful as a model animal with endocrinological defects in pituitary PRL and GH.

Correlation between Retention and Hydrocarbonaceous Area of Non-protein Alicyclic α-Amino Acids in Reversed-phase HPLC.

The retention by reversed-phase HPLC of a homologous series of aliphatic amino acids increased as the size of the apolar moiety increased. By use of the hydrocarbonaceous surface area as the structural descriptor, we found that cyclic amino acids had higher retention indices than the corresponding linear homologues. The retention difference between ring and chain compounds probably arises from the entropy effects.

Quantitative and morphological studies on the influence of zinc deficiency on the liver of pregnant rats.

The effect of zinc deficiency on trace metals in the liver, spleen, kidney, pancreas and duodenum was investigated in the control and zinc-deficient rats at 17 days and 20 days of pregnancy. Zinc-deficient rats fell into limosis after 5 days of pregnancy. The contents of zinc, iron, copper and manganese in the maternal tissues were measured by colorimetry with 5NPPF. The morphological changes of the liver were observed by light and electron microscopy. The contents of zinc in the pancreas and duodenum were less in the zinc-deficient group than in the control at both 17 days and 20 days of pregnancy. The contents of the copper and manganese in the liver, kidney, pancreas, duodenum and spleen, however, were not significantly different from the control. The contents of iron in the liver, spleen and kidney in the deficient group increased greatly at 17 days and 20 days of pregnancy compared with the control group. The combination rate of transferrin with iron also increased significantly in the deficient group. Staining with Berlin blue or Turnbull's blue showed intense reaction to iron around the interlobular connective tissue of the liver in the zinc deficient group at 20 days of pregnancy. Ultrastructurally, the liver of the zinc-deficient rats showed the decrease of glycogen granules and increase of lipid-like granules and lysosomes with various sizes and electron densities. These findings suggest that zinc deficiency causes the increase of iron contents in the various organs during pregnancy, and that there is an intimate interrelationship between zinc and iron in the metabolism of iron during pregnancy.

Scanning and transmission electron microscopy of biodegradable microspheres (DSM) in blood vessels.

A type of biodegradable microsphere (DSM), approximately 45 microns in diameter, made of polymerized potato starch (Pharmacia, Sweden) was intravenously injected into rats to observe the state of DSM in small blood vessels in the kidney and liver at the electron microscopic level. Prior to their digestion with amylase, individual DSM changed their round shape to an irregularly folded one to occupy almost the whole area of the lumen. At the transmission electron microscopic level, DSM were impregnated with colloidal iron and were easily identified. Interaction of the iron labelled DSM with the surface of endothelial cells was unexpectedly loose and no adherence or fusion of this surface was observed. The starch substance was not visible in the pinocytotic vesicles of the endothelium. These findings suggest the independent profile of DSM in situ.

[Influence of body weight and population density on the number of ova shed in the superovulation-treated adult rat].

The effects of body weight and population density on the number of ova shed were examined in the superovulation-treated adult rat in order to clarify the variation of the number of ova shed. Twelve-week-old female rats, weighing 182-277 g, were injected with 40 i.u. of PMSG on metestrus, with sodium pentobarbital on diestrus ("critical period"), and with 40 i.u. of hCG, on estrus. Twenty hours after the injection, the number of ova shed in an ampulla was observed. There was no correlation between body weight and the number of ova shed (r = 0.004). The rats were kept in at three, six or nine rats per cage (260 X 380 X 180 mm) from weaning at 30 days old to 12 weeks, with superovulation treatment. There was no significant difference in the number of ova shed among the various population densities.

The influence of age on the number of non-atretic follicles classified based on follicular size in the rat ovary.

The age-related changes in the number of follicles classified according to size were investigated. The number of large follicles expected to ovulate spontaneously was not influenced by age, though the number of small follicles 250-549 microns in diameter, recovered by PMSG and hCG, was not constant. There was no relationship between the number of large follicles and small ones. This study suggests that the variation in ovum count after superovulation depending on age may be based on the variation in the number of small follicles.

[Effect of sodium pentobarbital on the number of ova shed in superovulation treated adult rats: relationship between the number of ova shed and dose of GTHs].

A new treatment for inducing superovulation in adult rats was examined. Virgin Wistar-Imamichi rats at 11 weeks of age were pretreated with PMSG on the metestrus day. Then, the animals were anesthetized with sodium pentobarbital on the expected estrus day. The maximum number of ova shed in the animals thus treated was 100 with a combination of 40 I. U. of PMSG and hCG. If the treatment with sodium pentobarbital was omitted, the maximum number remained at 43 with different combinations of PMSG and hCG dosages.