Estrogen and progesterone dose-dependently reduce disruptive effects of restraint on lordosis behavior

Ovariectomized rats were hormonally primed with various doses of estradiol benzoate (EB; 0.5-10 microg) in combination with various doses of progesterone (2.5-500 microg) to induce sexual receptivity. Females were then subjected to 5 min restraint and the effect on lordosis behavior was monitored for the next 30 min. Such mild stress has been previously shown to transiently reduce lordosis behavior of ovariectomized females hormonally primed only with 10 microg EB. In the current study, doses of progesterone of 25 microg or more in combination with 10 microg EB reduced the effects of restraint. Also priming doses of EB from 4.0 to 10 microg in combination with 250 microg progesterone prevented the lordosis-inhibiting effects of restraint. These findings reinforce prior observations of the dose-dependency of both estrogen and progesterone in the facilitation of lordosis behavior and introduce the female's lordosis response to mild restraint as a potentially useful index of the female's response to stress.     

Appropriate NR1-NR1 disulfide-linked homodimer formation is requisite for efficient expression of functional, cell surface N-methyl-D-aspartate NR1/NR2 receptors

A c-Myc epitope-tagged N-methyl-D-aspartate receptor NR1-2a subunit was generated, NR1-2a(c-Myc), where the tag was inserted after amino acid 81. NR1-2a(c-Myc) /NR2A receptors when expressed in mammalian cells are not trafficked to the cell surface nor do they yield cell cytotoxicity post-transfection. NR1-2a(c-Myc) was, however, shown to assemble with NR2A subunits by immunoprecipitation and [(3)H]MK801 radioligand binding assays. Immunoblots of cells co-transfected with wild-type NR1-2a/NR2A subunits yielded two NR1-2a immunoreactive species with molecular masses of 115 and 226 kDa. Two-dimensional electrophoresis under non-reducing and reducing conditions revealed that the 226-kDa band contained disulfide-linked NR1-2a subunits. Only the 115-kDa NR1-2a species was detected for NR1-2a(c-Myc)/NR2A. The c-Myc epitope is inserted adjacent to cysteine 79 of the NR1-2a subunit; therefore, it is possible that the tag may prevent the formation of NR1 disulfide bridges. A series of cysteine --> alanine NR1-2a mutants was generated, and the NR1-2a mutants were co-expressed with NR2A or NR2B subunits in mammalian cells and characterized with respect to cell surface expression, cell cytotoxicity post-transfection, co-association by immunoprecipitation, and immunoblotting following SDS-PAGE under both reducing and non-reducing conditions. When co-expressed with NR2A in mammalian cells, NR1-2a(C79A)/NR2A displayed similar properties to NR1-2a(c-Myc)/NR2A in that the 226-kDa NR1 immunoreactive species was not detectable, and trafficking to the cell surface was impaired compared with wild-type NR1/NR2 receptors. These results provide the first biochemical evidence for the formation of NR1-NR1 intersubunit disulfide-linked homodimers involving cysteine 79. They suggest that disulfide bridging and structural integrity within the NR1 N-terminal domain is requisite for cell surface N-methyl-D-aspartate receptor expression.     

Regulation of alpha 2(I) collagen expression in stellate cells by retinoic acid and retinoid X receptors through interactions with their cofactors

Retinoic acid (RA) suppresses alpha 2(I) collagen expression in hepatic stellate cells through the binding of retinoic acid receptor beta (RAR beta) and retinoid X receptor alpha (RXR alpha) to RA response elements (RAREs) in the alpha 2(I) collagen promoter. This study determined the influence of coactivators and corepressors to RAR beta and RXR alpha on the regulation of the alpha 2(I) collagen promoter. The coactivators, steroid receptor coactivator-1 (SRC-1) and growth hormone receptor interacting protein-1 (GRIP-1), enhanced, while the nuclear receptor corepressor (N-CoR) abolished the inhibitory effect of RAR beta and RXR alpha on the promoter activity. In the presence of RA, the coactivators SRC-1 and GRIP-1 formed complexes with RAR beta and RXR alpha which are bound to an oligonucleotide specifying a RARE site in the promoter. In conclusion, this study shows that in the presence of retinoic acid, the coactivators SRC-1 and GRIP-1 augment, while the corepressor N-CoR abolishes, the suppressive effects of RAR beta and RXR alpha on alpha 2(I) collagen promoter activity.     

Recognition of an unnatural difluorophenyl nucleotide by uracil DNA glycosylase

The DNA repair enzyme uracil DNA glycosylase (UDG) utilizes base flipping to recognize and remove unwanted uracil bases from the genome but does not react with its structural congener, thymine, which differs by a single methyl group. Two factors that determine whether an enzyme flips a base from the duplex are its shape and hydrogen bonding properties. To probe the role of these factors in uracil recognition by UDG, we have synthesized a DNA duplex that contains a single difluorophenyl (F) nucleotide analogue that is an excellent isostere of uracil but possesses no hydrogen bond donor or acceptor groups. By using binding affinity measurements, solution (19)F NMR, and solid state (31)P[(19)F] rotational-echo double-resonance (REDOR) NMR measurements, we establish that UDG partially unstacks F from the duplex. However, due to the lack of hydrogen bonding groups that are required to support an open-to-closed conformational transition in UDG, F cannot stably dock in the UDG active site. We propose that F attains a metastable unstacked state that mimics a previously detected intermediate on the uracil-flipping pathway and suggest structural models of the metastable state that are consistent with the REDOR NMR measurements.     

Catalase-peroxidases (KatG) exhibit NADH oxidase activity

Catalase-peroxidases (KatG) produced by Burkholderia pseudomallei, Escherichia coli, and Mycobacterium tuberculosis catalyze the oxidation of NADH to form NAD+ and either H2O2 or superoxide radical depending on pH. The NADH oxidase reaction requires molecular oxygen, does not require hydrogen peroxide, is not inhibited by superoxide dismutase or catalase, and has a pH optimum of 8.75, clearly differentiating it from the peroxidase and catalase reactions with pH optima of 5.5 and 6.5, respectively, and from the NADH peroxidase-oxidase reaction of horseradish peroxidase. B. pseudomallei KatG has a relatively high affinity for NADH (Km=12 microm), but the oxidase reaction is slow (kcat=0.54 min(-1)) compared with the peroxidase and catalase reactions. The catalase-peroxidases also catalyze the hydrazinolysis of isonicotinic acid hydrazide (INH) in an oxygen- and H2O2-independent reaction, and KatG-dependent radical generation from a mixture of NADH and INH is two to three times faster than the combined rates of separate reactions with NADH and INH alone. The major products from the coupled reaction, identified by high pressure liquid chromatography fractionation and mass spectrometry, are NAD+ and isonicotinoyl-NAD, the activated form of isoniazid that inhibits mycolic acid synthesis in M. tuberculosis. Isonicotinoyl-NAD synthesis from a mixture of NAD+ and INH is KatG-dependent and is activated by manganese ion. M. tuberculosis KatG catalyzes isonicotinoyl-NAD formation from NAD+ and INH more efficiently than B. pseudomallei KatG.     

Identification of a novel NF-kappaB-binding site with regulation of the murine alpha2(I) collagen promoter

Hepatic fibrosis is due to the increased synthesis and deposition of type I collagen. Acetaldehyde activates type I collagen promoters. Nuclear factor kappaB (NF-kappaB) was previously shown to inhibit expression of murine alpha(1)(I) and human alpha(2)(I) collagen promoters. The present study identifies binding of NF-kappaB, present in nuclear extracts of stellate cells, to a region between -553 and -537 of the murine alpha(2)(I) collagen promoter. The NF-kappaB (p65) expression vector inhibited promoter activity. Mutation of the promoter at the NF-kappaB-binding site increased basal promoter activity and abrogated the activating and inhibitory effects of transforming growth factor beta and tumor necrosis factor alpha, respectively, on promoter activity. Acetaldehyde increased IkappaB-alpha kinase activity and phosphorylated IkappaB-alpha, NF-kappaB nuclear protein, and its binding to the promoter. However, the activating effect of acetaldehyde was not affected by the mutation of the promoter. In conclusion, although acetaldehyde increases the binding of NF-kappaB to the murine alpha(2)(I) collagen promoter, this binding does not mediate the activating effect of acetaldehyde on promoter activity. The effects of acetaldehyde in increasing the translocation of NF-kappaB to the nucleus with increased DNA binding activity may be important in mediating the effects of acetaldehyde on other genes.     

Morphology and histochemistry of the peripheral olfactory organ in the round goby,Neogobius melanostomus (Teleostei: Gobiidae)

This first comprehensive study of the peripheral olfactory organ from a representative of the large and economically important order of teleost fishes, the Perciformes, shows a compact structure with olfactory sensory neurons distributed widely throughout the olfactory chamber. The spatial organization of the nasal cavity in the bottom-dwelling round goby (Gobiidae, Neogobius melanostomus) was examined using impression material injection, immunocytochemistry, and transmission electron microscopy. The olfactory chamber contains a single olfactory lamella; prominent dorsocaudal lachrymal and ethmoidal accessory nasal sacs are situated ventrocaudal to the chamber. The location of the olfactory mucosa within the olfactory chamber is novel for teleost fish, as it extends beyond the ventral surface to the lateral and dorsal regions. Microvillar olfactory sensory neurons and ciliated olfactory sensory neurons were identified by transmission electron microscopy and the spatial distribution of these two cell types was assessed through immunocytochemistry against olfactory receptor coupled G-proteins. Both G(alphaolf)-immunoreactive ciliated olfactory sensory neurons and the G(alphao)-immunoreactive microvillar form were located throughout the olfactory epithelium. Ciliated crypt cells were G(alphao) immunoreactive and were found throughout the olfactory epithelium of some specimens. The widespread occurrence of olfactory sensory neurons in the olfactory chamber supports the idea that olfactory signaling is important to the survival of the round goby. The prominence of the lachrymal and ethmoidal accessory nasal sacs indicates the capacity to regulate the flow of odorant molecules over the sensory surface of the olfactory sensory neurons, possibly through a pump-like mechanism driven by opercular activity associated with gill ventilation.     

Deoxycytidine glyoxal: lesion induction and evidence of repair following vitamin C supplementation in vivo

Oxidative DNA damage is postulated to be involved in carcinogenesis, and as a consequence, dietary antioxidants have received much interest. A recent report indicates that vitamin C facilitates the decomposition of hydroperoxides in vitro, generating reactive aldehydes. We present evidence for the in vivo generation of glyoxal, an established product of lipid peroxidation, glucose/ascorbate autoxidation, or free radical attack of deoxyribose, following supplementation of volunteers with 400 mg/d vitamin C. Utilizing a monoclonal antibody to a deoxycytidine-glyoxal adduct (gdC), we measured DNA lesion levels in peripheral blood mononuclear cells. Supplementation resulted in significant (p =.001) increases in gdC levels at weeks 11, 16, and 21, with corresponding increases in plasma malondialdehyde levels and, coupled with previous findings, is strongly suggestive of a pro-oxidative effect. However, continued supplementation revealed a highly significant (p =.0001) reduction in gdC levels. Simultaneous analysis of cyclobutane thymine dimers revealed no increase upon supplementation but, as with gdC, levels decreased. Although no single mechanism is identified, our data demonstrate a pro-oxidant event in the generation of reactive aldehydes following vitamin C supplementation in vivo. These results are also consistent with our hypothesis for a role of vitamin C in an adaptive/repair response and indicate that nucleotide excision repair specifically may be affected.     

Carbon monoxide neurotransmission activated by CK2 phosphorylation of heme oxygenase-2

Carbon monoxide (CO) is a putative gaseous neurotransmitter that lacks vesicular storage and must be synthesized rapidly following neuronal depolarization. We show that the biosynthetic enzyme for CO, heme oxygenase-2 (HO2), is activated during neuronal stimulation by phosphorylation by CK2 (formerly casein kinase 2). Phorbol ester treatment of hippocampal cultures results in the phosphorylation and activation of HO2 by CK2, implicating protein kinase C (PKC) in CK2 stimulation. Odorant treatment of olfactory receptor neurons augments HO2 phosphorylation and activity as well as cyclic guanosine monophosphate (cGMP) levels, with all of these effects selectively blocked by CK2 inhibitors. Likewise, CO-mediated nonadrenergic, noncholinergic (NANC) relaxation of the internal anal sphincter requires CK2 activity. Our findings provide a molecular mechanism for the rapid neuronal activation of CO biosynthesis, as required for a gaseous neurotransmitter.     

Genetic constraints on floral evolution in a sexually dimorphic plant revealed by artificial selection

Sexual dimorphism is one of the most widespread and recognizable patterns of phenotypic variation in the biotic world. Sexual dimorphism in floral display is striking in the dioecious plant Silene latifolia, with males making many, small flowers compared to females. We investigated this dimorphism via artificial selection on two populations to determine whether genetic variation exists within populations for flower size and the extent of the between-sex correlation, whether a flower size and number trade-off exists within each sex, and whether pollen and ovule production vary with flower size. We selected for decreased flower size (calyx width) in females and increased flower size in males and measured the response to selection in size and correlated responses in flower dry mass, flower number, and pollen or ovule number per flower. Four bouts of selection in each of two selection programs were performed, for a total of three selection lines to decrease size, three to increase it, and two control lines. Flower size always significantly responded to selection and we always found a significant correlated response in the sex not under selection. Selection decreased but did not eliminate the sexual dimorphism in flower dry mass and number. A negative relationship between flower size and number within each sex was revealed. Whereas ovule number showed a significant correlated response to selection on flower size, pollen number did not. Our results indicate that although substantial additive genetic variation for flower size exists, the high between-sex genetic correlation would likely constrain flower size from becoming more sexually dimorphic. Furthermore, floral display within each sex is constrained by a flower size and number trade-off. Given this trade-off and lack of variation in pollen production with flower size, we suggest that sexual dimorphism evolved via sexual selection to increase flower number in males but not females.