Using c-Fos/c-Jun as hetero-dimer interaction model to optimize donor to acceptor concentration ratio range for three-filter fluorescence resonance energy transfer (FRET) measurement.

Sensitized emission FRET detection method based on three-filter fluorescence microscopy is widely used and more suitable for live cell FRET imaging and dynamic protein-protein interaction analysis. But when it is applied to detect two proteins interaction in living cells, this intensity-based detection method is complicated by many experimental factors such as spectral crosstalk and spectral bleed-through and variable donor to acceptor concentration ratio. There are several FRET algorithms developed recently to correct those factors in order to quantitatively gauge and compare FRET signals between different experimental groups. But the algorithms are often difficult to choose when they are applied to certain experiments. In this research, we use c-Fos/c-Jun as a simple hetero-dimer interaction model to quantitatively detect and compare the FRET signals based on the following widely used sensitized emission FRET algorithms: N(FRET) , FRET(N) , FR, FRET(R) , E(app) and E(EFF) . We optimized the donor to acceptor concentration ratio range for the above FRET algorithms and facilitate their use in accurate FRET signal determination based on the three-filter FRET microscopy.

Gene transfer of calcitonin gene-related peptide suppresses development of allograft vasculopathy.

OBJECTIVE: To explore suppression of allograft vasculopathy by transfer of the calcitonin gene-related peptide (CGRP). METHODS: The descending thoracic aortas from Lewis rats were grafted to the abdominal aortas of F344 rats, and the rats were randomized into 2 groups. A gene construct containing sequences from the adenoviral oncoprotein, the CGRP, and the enhanced green fluorescent protein was transferred into 1 group, and the sequences for the adenoviral oncoprotein and enhanced green fluorescent protein were transferred into a control group. Specimens were harvested at 4 and 8 weeks. Gene transfer was confirmed at fluorescence microscopy of frozen tissue sections, and expression was measured using reverse transcriptase-polymerase chain reaction. We determined the locations and levels of vascular cell adhesion molecule-1 (VCAM-1) and inducible nitric oxide synthase (iNOS) at immunohistochemistry and measured apoptosis. RESULTS: The CGRP gene was expressed only in the CGRP group at 4 weeks. The vascular luminal occlusion score in the CGRP group was lower than in the control group. The apoptotic index of the CGRP group was lower than in the control group only at 4 weeks. The VCAM-1 immunohistochemistry score in the CGRP group was lower than in the control group; however, the iNOS immunohistochemistry score in the CGRP group was lower than in the control group in the intima only at 4 weeks. CONCLUSION: The expression of CGRP effectively suppressed the development of allograft vasculopathy and encroachment by lymphocytes and inflammatory cells. This reduced the levels of VCAM-1 to inhibit apoptosis induced by iNOS; thus, the tissue of the allografted vessel was protected and rejection was averted.

mRNA expression of gonadotropin-releasing hormones (GnRHs) and GnRH receptor in goldfish.

In goldfish (Carassius auratus), two distinct forms of gonadotropin-releasing hormone (GnRH), namely, salmon GnRH (sGnRH) and chicken GnRH-II (cGnRH-II), have been identified in the brain using chromatographic, immunological, and molecular cloning approaches. These two native GnRHs act on specific receptors in the anterior pituitary to stimulate the synthesis and release of gonadotropins and growth hormone in goldfish. To evaluate the potential roles of sGnRH and cGnRH-II in both neural and reproductive tissues in goldfish, we studied the mRNA expression of sGnRH, cGnRH-II, and GnRH receptor (GnRH-R) in discrete brain areas, pituitary, ovary, and testis by a combined reverse transcription-polymerase chain reaction (RT-PCR) and Southern blot analysis. Total RNA was extracted from various tissues of sexually recrudescent male and female goldfish and RT-PCR was performed with primers specific for GnRH-R complementary DNA (cDNA), sGnRH cDNA, cGnRH-II cDNA-1, and cDNA-2. Results showed that GnRHs and GnRH-R mRNAs are differentially distributed in the brain. In the goldfish brain, sGnRH mRNA was predominantly expressed in the forebrain areas (olfactory bulb, telencephalon, and hypothalamus) whereas cGnRH-II mRNA-1 were expressed in all brain areas including olfactory bulbs and optic tectum-thalamus. The expression level of cGnRH-II mRNA-2 was much lower than that of cGnRH-II mRNA-1 in the brain. On the other hand, GnRH-R mRNA was expressed in all brain regions and pituitary. In the ovary and testis, GnRH-R mRNA, sGnRH mRNA, and cGnRH-II mRNA-1, but not cGnRH-II mRNA-2, are expressed. Sequence analysis of the PCR products showed that nucleotide sequences of GnRH-R in gonads are identical with that in the brain and pituitary. The coexistence of GnRHs and GnRH-R mRNAs in both neural and gonadal tissues supports the notion that sGnRH and cGnRH-II may act as neurotransmitters and/or neuromodulators in the brain and as autocrine and/or paracrine hormones in gonadal tissues in addition to their established neuroendocrine roles at the pituitary of goldfish.

Evolution of neuroendocrine peptide systems: gonadotropin-releasing hormone and somatostatin.

Nine vertebrate and two protochordate gonadotropin-releasing hormone (GnRH) decapeptides have been identified and sequenced. Multiple molecular forms of GnRH peptide were present in the brain of most species examined, and cGnRH-II generally coexists with one or more GnRH forms in all the major vertebrate groups. The presence of multiple GnRH forms has been further confirmed by the deduced GnRH peptide structure from cDNA and/or gene sequences in several teleost species and tree shrew. High conservation of the primary structure of GnRH decapeptides and the overall structure of GnRH genes and precursors suggests that they are derived from a common ancestor. Somatostatin (SRIF) is a phylogenetically ancient, multigene family of peptides. A tetradecapeptide, SRIF (SRIF14) has been conserved, with the same amino acid sequence, in representative species of all classes of vertebrate. Four molecular variants of SRIF14 have been identified. SRIF14 is processed from preprosomatostatin-I, which contains SRIF14 at its C-terminus; preprosomatostatin-I is also processed to SRIF28 in mammals and SRIF26 in bowfin. Teleost fish possess a second somatostatin precursor, preprosomatostatin-II, containing [Tyr7, Gly10]-SRIF14 at the C-terminus, that is mainly processed into large forms of SRIF.

Cloning and expression pattern of a second [His5Trp7Tyr8]gonadotropin-releasing hormone (chicken GnRH-H-II) mRNA in goldfish: evidence for two distinct genes.

Complementary DNAs (cDNAs) encoding [Trp7Leu8]GnRH (sGnRH) and [His5Trp7Tyr8]GnRH (cGnRH-II) peptides have been isolated from the brain of goldfish (X. W. Lin and R. E. Peter, 1996, Gen. Comp. Endocrinol. 101, 282-296). In the present study we report the isolation of a second cDNA encoding cGnRH-II peptide in the brain of goldfish using reverse transcription (RT) and rapid amplification of cDNA ends. There is an overall 79.7% nucleotide sequence similarity between the two cGnRH-II cDNAs, with 65.3, 91.2, and 76.3% similarity between the 5'-untranslated regions, coding regions, and 3'-untranslated regions, respectively, of the two cGnRH-II cDNAs. Comparison of the two cGnRH-II precursors shows 87.2% amino acid similarity. The presence of two cGnRH-II genes was confirmed by the sequence analysis of the introns between exon II and exon III of the two cGnRH-II genes. Results indicate that the intron of the two cGnRH-II genes shows a high divergence in size and sequence, but contains the same splice junction. Expression of the two cGnRH-II mRNAs was detected by RT-polymerase chain reaction assay and Southern blot analysis in all five grossly dissected brain areas, olfactory bulbs and tracts, telencephalon, hypothalamus, optic tectum-thalamus, and posterior brain. However, there was a difference in apparent intensity of hybridization signal for the two cGnRH-II mRNAs in all brain areas, suggesting a difference of expression levels. sGnRH mRNA was detected in the olfactory bulbs, telencephalon, and hypothalamus, but not in midbrain and posterior brain areas. The present finding of duplicate cDNAs and genes for cGnRH-II in goldfish is in agreement with the recent tetraploidization in this species.

Expression of salmon gonadotropin-releasing hormone (GnRH) and chicken GnRH-II precursor messenger ribonucleic acids in the brain and ovary of goldfish.

The complementary DNAs (cDNA) encoding the [Trp7Leu8]gonadotropin-releasing hormone (salmon GnRH; sGnRH) precursor and the [His5Trp7Tyr8]GnRH (chicken GnRH-II; cGnRH-II) precursor of the goldfish brain were isolated and sequenced using reverse transcription and rapid amplification of cDNA ends (RACE). The sGnRH precursor cDNA consists of 540 bp, including an open reading frame of 282 bp, and the cGnRH-II precursor cDNA consists of 682 bp, including an open reading frame of 258 bp. The 94 amino acid-long goldfish sGnRH precursor and 86 amino acid-long goldfish cGnRH-II precursor have the same molecular architecture as GnRH precursors identified to date in other vertebrate species. Using two sets of primers designed to be sense and antisense to the goldfish brain sGnRH precursor cDNA sequence, reverse transcription-polymerase chain reaction (RT-PCR) amplification of total RNA from brain and ovary at gonadal recrudescent, mature ( = prespawning), and postovulatory stages resulted in two predicted sizes of PCR products. The intensities of staining signals of ethidium bromide were similar between brain and ovary samples. The same RT-PCRs were carried out with two sets of primers for cGnRH-II precursor cDNA, resulting in two PCR products of predicted size; however, the ethidium bromide staining signals are much weaker for products amplified from ovarian cDNA than that from brain cDNA. Restriction enzyme analysis verified the expected RT-PCR products. Sequence analysis of ovarian sGnRH precursor cDNA generated by RACE of total RNA from recrudescent ovarian tissue revealed the identical sequence to that of the brain sGnRH cDNA. Northern blot analysis detected a single mRNA transcript of approximately 650 bases for the sGnRH precursor in both the brain and ovary, and 750 bases for the cGnRH-II precursor in the brain. These results demonstrate that two forms of GnRH precursor (sGnRH and cGnRH-II) mRNA are expressed in goldfish brain tissue and that the sGnRH transcript and a low level of cGnRH-II transcript are also expressed in the goldfish ovary.

Seasonal variations in gonadotropin responsiveness, self-priming, and desensitization to GnRH peptides in the common carp pituitary in vitro.

Seasonal variations of the GtH release response to salmon GnRH (sGnRH) and [D-Arg6,Pro9NEt]-sGnRH (sGnRH-A) were investigated in female common carp at different stages of the reproductive cycle using perifused pituitary fragments. The responsiveness to sGnRH and sGnRH-A varied seasonally in common carp pituitaries in vitro, with the greatest GtH release response in pituitaries from sexually mature (preovulatory) fish compared to pituitaries from sexually regressed fish. The magnitude of this seasonal change in the GtH release response was greater for sGnRH-A than for sGnRH, and sGnRH-A has a higher potency than sGnRH, particularly in pituitaries from sexually mature fish. Desensitization of perifused pituitary fragments to sGnRH and sGnRH-A, and a self-priming effect of sGnRH-A on the GtH release response, caused by repeated pulse administrations of the GnRH peptides, varied with the stage of reproductive cycle of the common carp. Using pituitaries from sexually regressed female common carp, desensitization occurred only when a high dose of sGnRH or sGnRH-A was given as repeated pulses at short time intervals, and no self-priming was observed by repeated administrations of sGnRH and sGnRH-A. Using pituitaries from sexually mature female common carp, desensitization occurred when a high dose of sGnRH and both high and low dosages of sGnRH-A were given as repeated pulses at short time intervals. Self-priming, largely due to the increase in basal GtH levels, occurred in response to repeated pulses of low dosages of sGnRH-A given at long intervals.

Growth hormone and gonadotropin secretion in the common carp (Cyprinus carpio L.): in vitro interactions of gonadotropin-releasing hormone, somatostatin, and the dopamine agonist apomorphine.

The effects of salmon gonadotropin-releasing hormone (sGnRH) and the superactive agonist [D-Arg6, Pro9NEt]-sGnRH (sGnRH-A) on growth hormone (GH) and gonadotropin (GtH) release were examined using a perifusion system for pituitary fragments of the common carp (Cyprinus carpio). Perifusion of 2-min pulses of different concentrations of sGnRH or sGnRH-A stimulated a rapid and dose-dependent increase in GH release: ED50 values for sGnRH and sGnRH-A in stimulating GH release were 2.8 +/- 0.7 and 0.5 +/- 0.1 nM, respectively, indicating that the superactivity of sGnRH-A for stimulation of GtH release also applies in induction of GH release. Exposure of the pituitary fragments to 10 nM sGnRH or sGnRH-A alone resulted in increases in GH and GtH release on a similar temporal course. Apomorphine (10, 100, and 1000 nM) significantly inhibited basal and GnRH-induced GtH release in a dose-dependent manner and significantly stimulated basal GH release; however, APO did not enhance GnRH-induced GH release. Somatostatin (100 nM) significantly blocked basal release and 10 nM sGnRH- and sGnRH-A-induced GH release, but was ineffective on GtH release. Treatment with somatostatin (100 nM) in combination with apomorphine (100 nM) caused an increase in sGnRH-induced GH release compared to treatment with somatostatin alone; whereas, on GtH there was a significant decrease in basal and GnRH-induced levels, compared to treatment with somatostatin alone. These results indicate that GH release in common carp is regulated by somatostatin as GH release inhibitor. sGnRH and sGnRH-A act as GH-releasing factors; the mechanisms by which GnRH stimulates GH and GtH secretion are independent. The dopamine agonist apomorphine stimulates GH release and inhibits GtH release directly at the pituitary level.

[The transplacental effect of methylmercury on the chromosome of embryo liver cells in rat].

The effect of methylmercury on the chromosome of embryo liver cells of rat was studied in vivo. The results showed that the rate of chromosome aberration both of embryo liver cells and maternal bone marrow cells increased linearly with the dose of methylmercury. The linear equations were as follows: Yembryo liver cell = 10.30 + 2.54D (r = 0.9573, P less than 0.01) Ymarrow cell = 2.34 + 0.71D (r = 0.9782, P less than 0.01).