ABSTRACT
Experimental and clinical data suggest that the Ginkgo biloba standardized extract EGb 761® exerts beneficial effects in conditions which are associated with impaired cognitive function. However, the neurochemical correlates of these memory enhancing effects are not yet fully clarified. The aim of this study was to examine the effect of repeated oral administration of EGb 761® and some of its characteristic constituents on extracellular levels of dopamine (DA), noradrenaline (NA), serotonin (5-HT), acetylcholine (ACh) and the metabolites 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA) in the medial prefrontal cortex (mPFC) of awake rats by use of in vivo microdialysis technique. Subacute (14 days, once daily), but not acute, oral treatment with EGb 761® (100 and 300 mg/kg) or the flavonoid fraction, which represents about 24% of the whole extract caused a significant and dose-dependent increase in extracellular DA levels in the mPFC. Repeated administration of EGb 761® also caused a modest but significant increase in the NA levels, whereas the concentrations of 5-HT and those of the metabolites DOPAC, HVA and 5-HIAA were not affected. The same treatment regimen was used in a subsequent study with the aim of investigating the effects of two Ginkgo-specific acylated flavonols, 3-O-(2''-O-(6'''-O-(p-hydroxy-trans-cinnamoyl)-ß-D-glucosyl)-α-L-rhamnosyl)quercetin (Q-ag) and 3-O-(2''-O-(6'''-O-(p-hydroxy-trans-cinnamoyl)-ß-D-glucosyl)-α-L-rhamnosyl)kaempferol (K-ag). Both compounds together represent about 4.5% of the whole extract. Repeated oral treatment with Q-ag (10 mg/kg) for 14 days caused a significant increase in extracellular DA levels of 159% and extracellular acetylcholine (ACh) levels of 151% compared to controls. Similarly, administration of K-ag (10 mg/kg) induced a significant rise of DA levels to 142% and ACh levels to 165% of controls, whereas treatment with isorhamnetin, an O-methylated aglycon component of EGb 761® flavonol glycosides had no effect. None of the tested flavonoids had a significant effect on extracellular DOPAC and HVA levels. The present findings provide evidence that the subacute treatment with EGb 761® and its flavonol constituents increases DA and ACh release in the rat mPFC, and suggest that the two Ginkgo-specific acylated flavonol glycosides Q-ag and K-ag are active constituents contributing to these effects. As seen for isorhamnetin, the effect on neurotransmitter levels seems not to be a general effect of flavonols but rather to be a specific action of acylated flavonol glycosides which are present in EGb 761®. The direct involvement of these two flavonol derivatives in the increase of dopaminergic and cholinergic neurotransmission in the prefrontal cortex may be one of the underlying mechanisms behind the reported effects of EGb 761® on the improvement of cognitive function.
Subject(s)
Acetylcholine/analysis , Cognition/drug effects , Dopamine/analysis , Plant Extracts/pharmacology , Prefrontal Cortex/drug effects , 3,4-Dihydroxyphenylacetic Acid/analysis , Animals , Dose-Response Relationship, Drug , Ginkgo biloba , Homovanillic Acid/analysis , Hydroxyindoleacetic Acid/analysis , Norepinephrine/analysis , Prefrontal Cortex/chemistry , Rats , Serotonin/analysisABSTRACT
AIM: To develop a sensitive and selective liquid-chromatographic method for the determination of histamine in microdialysis samples from guinea pig skin following allergenic provocation. METHODS: The novel fluorescence derivatization method is based on an intramolecular excimer-forming reaction between 2 amino moieties of histamine and 2 molecules of 4-(1-pyrene)butanoyl chloride (PBC) yielding the corresponding dipyrene-labeled derivative. RESULTS: The PBC derivative of histamine was separated within 20 min, and the detection limit (signal-to-noise ratio = 3) of histamine was 0.6 fmol/20 µl volume injected. The basal extracellular levels of histamine in guinea pig skin microdialysates were 20.6 ± 1.7 fmol/10 µl. Subcutaneous administration of histamine liberator compound 48/80 (3 mg/kg) increased the extracellular histamine levels in the skin dialysates by about 860%, whereas ovalbumin challenge (2 mg/kg i.v.) in the sensitized guinea pigs increased the extracellular histamine levels by about 3,030%. CONCLUSION: The novel technique for histamine determination in microdialysis samples from the guinea pig skin may be utilized in preclinical research of antihistaminergic drugs and evaluation of allergenic properties of various dermal preparations such as transdermal drug delivery systems.
Subject(s)
Chromatography, High Pressure Liquid/methods , Histamine/analysis , Microdialysis/methods , Spectrometry, Fluorescence/methods , Animals , Guinea Pigs , Male , Ovalbumin/immunology , Skin/metabolism , p-Methoxy-N-methylphenethylamine/immunologyABSTRACT
In order to study the regulation of the growth differentiation factor-9 (gdf9) gene in a primitive teleost with semelparous life history, we cloned a cDNA encoding shortfinned eel Gdf9, expressed a partial peptide in Escherichia coli, and raised an antiserum to evaluate changes in Gdf9 expression during its pituitary homogenate-induced reproductive cycle. The effects of in vivo and in vitro exposure to the androgen 11-ketotestosterone (11-KT), known to affect previtellogenic (PV) oocyte growth, were also determined. Furthermore, we investigated whether Gdf9 expression was metabolically gated by treating PV fish with recombinant GH in vivo. Immunoreactive proteins of ca. 52 and 55 kDa were identified by western blot analysis. Gdf9 message and protein were most abundant in PV oocytes, and peaked slightly earlier for mRNA than for protein. Captivity resulted in reduced gdf9 mRNA levels, which were restored following pituitary homogenate treatment. As oocytes progressed through induced oogenesis, Gdf9 expression decreased. Neither 11-KT nor GH treatment affected gdf9 mRNA levels in PV fish, although GH could partially restore handling- or captivity-induced decreases in gdf9 mRNA levels. Semelparous eels thus show an expression pattern of Gdf9 during oogenesis that is similar to that seen in other vertebrates, that appears responsive to handling or captivity stress, and whose control remains to be elucidated.
Subject(s)
Androgens/pharmacology , Anguilla/physiology , Growth Differentiation Factor 9/metabolism , Growth Hormone/pharmacology , Ovary/metabolism , Reproduction , Testosterone/analogs & derivatives , Anguilla/growth & development , Anguilla/metabolism , Animals , Female , Fish Proteins/genetics , Fish Proteins/metabolism , Gene Expression Regulation, Developmental/drug effects , Growth Differentiation Factor 9/chemistry , Growth Differentiation Factor 9/genetics , Growth Differentiation Factor 9/isolation & purification , Growth Hormone/analogs & derivatives , Immunohistochemistry , In Situ Hybridization , Insulin-Like Growth Factor I/metabolism , Nucleic Acid Amplification Techniques , Oogenesis , Organ Specificity , Ovary/cytology , Ovary/drug effects , Ovary/growth & development , Phylogeny , RNA, Messenger/metabolism , Sequence Analysis, RNA , Stress, Physiological , Testosterone/pharmacologyABSTRACT
Normal migration of the gonadotrophin-releasing hormone (GnRH) neurones during early development, from the olfactory region to the hypothalamus, is crucial for reproductive development in all vertebrates. The establishment of the GnRH system includes tangential migration of GnRH perikarya as well as extension of GnRH fibres to various areas of the central nervous system (CNS). The exact spatio-temporal nature of this process, as well as the factors governing it, are not fully understood. We studied the development of the GnRH system and the effects of GnRH knockdown using a newly developed GnRH3:EGFP transgenic zebrafish line. We found that enhanced green fluorescent protein is specifically and robustly expressed in GnRH3 neurones and fibres. GnRH3 fibres in zebrafish began to extend as early as 26 h post-fertilisation and by 4-5 days post-fertilisation had developed into an extensive network reaching the optic tract, telencephalon, hypothalamus, midbrain tegmentum and hindbrain. GnRH3 fibres also innervated the retina and projected into the trunk via the spinal cord. GnRH3 perikarya were observed migrating along their own fibres from the olfactory region to the preoptic area (POA) via the terminal nerve ganglion and the ventral telencephalon. GnRH3 cells were also observed in the trigeminal ganglion. The establishment of the GnRH3 fibre network was disrupted by morpholino-modified antisense oligonucleotides directed against GnRH3 causing abnormal fibre development and pathfinding, as well as anomalous GnRH3 perikarya localisation. These findings support the hypothesis that GnRH3 neurones migrate from the olfactory region to the POA and caudal hypothalamus. Novel data regarding the early development of the GnRH3 fibre network in the CNS and beyond are described. Moreover we show, in vivo, that GnRH3 is an important factor regulating GnRH3 fibre pathfinding and neurone localisation in an autocrine fashion.
Subject(s)
Autocrine Communication/physiology , Cell Movement/genetics , Gonadotropin-Releasing Hormone/genetics , Gonadotropin-Releasing Hormone/physiology , Neurons/metabolism , Oligopeptides/genetics , Oligopeptides/physiology , Prosencephalon/embryology , Pyrrolidonecarboxylic Acid/analogs & derivatives , Zebrafish/embryology , Animals , Animals, Genetically Modified , Axons/metabolism , Embryo, Nonmammalian , Gonadotropin-Releasing Hormone/metabolism , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Oligopeptides/metabolism , Prosencephalon/metabolism , Pyrrolidonecarboxylic Acid/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Time Factors , Zebrafish/geneticsABSTRACT
Partial cDNAs encoding carbonyl reductase like 20beta-hydroxysteroid dehydrogenase (20beta-HSD) and P450 17alpha-hydroxylase/c17-20 lyase (CYP17) were isolated from the ovary of snake head murrel and they exhibited high sequence identity to the Nile tilapia and rainbow trout, respectively. A low transcript level of both 20beta-HSD and CYP17 were detected in pre-vitellogenic follicles, while the transcript level was high in full-grown immature follicles. In hCG-induced in vitro oocyte maturation, we found a significant increase in 20beta-HSD transcript level after 2 h. The CYP17 transcripts also showed a considerable increase following hCG-induction compared to saline-treated controls. On the other hand, Western blot analysis demonstrated no significant change in the CYP17 protein level during hCG-induced in vitro oocyte maturation. Taken together, we suggest that in addition to 20beta-HSD, the CYP17 might have a role in the shift in steroidogenesis during meiotic maturation of snake head murrel.
ABSTRACT
Differential display of mRNA was used to identify an upregulated gene in ovaries of artificially maturing Japanese eels (Anguilla japonica). Accordingly, mitochondrial (mt) cytochrome b, whose transcript levels increased from early to late vitellogenesis, was isolated, cloned and sequenced. Temporal trends in artificially maturing eels were compared with those in naturally and artificially maturing New Zealand eels (longfinned eel, Anguilla dieffenbachii; shortfinned eel, Anguilla australis) by Northern blot and in situ hybridization analysis to rule out any experimental artifacts. An increase in ovarian mt cytochrome b signals was seen when comparing immature and midvitellogenic longfinned eels, but not immature and early vitellogenic shortfinned eels, from the wild. Long-term captivity yielded reduced target mRNA levels, but abundance increased after hormonal induction of vitellogenesis. These results imply that the increase in mt cytochrome b mRNA levels during artificial maturation reflects natural development, although its onset appears to be brought forward during artificial maturation in the Japanese eel. It is suggested that increased mt cytochrome b mRNA levels result from both mitochondrial replication and increased transcription, and that they reflect the build-up of machinery for enhanced ATP-synthesis at some stage of oogenesis and/or early zygote development.
Subject(s)
Cytochromes b/genetics , Eels/physiology , Ovary/physiology , Sexual Maturation/physiology , Animals , Blotting, Northern , Cloning, Molecular , Energy Metabolism/physiology , Female , Fresh Water , Gene Expression/physiology , In Situ Hybridization , Japan , Mitochondria/physiology , New Zealand , Oogenesis/physiology , RNA, Messenger/metabolismABSTRACT
This report describes the structure of the 5'-flanking regions of both the CYP19A1 and A2 genes that were isolated from the genome of the zebrafish (Danio rerio). Consensus sequences of three cAMP-responsive elements (CRE), an aryl hydrocarbon-responsive element (AhR/Arnt), a steroidogenic factor 1 (SF-1) site, and a TATA box were observed in the 5'-flanking region of CYP19A1. In contrast, the 5'-flanking region of CYP19A2 was located upstream of an untranslated exon and possessed consensus sequences of a single CRE, an estrogen-responsive element (ERE), a peroxisome proliferator-activated receptor alpha/retinoid X receptor alpha heterodimer-responsive element (PPARalpha/RXRalpha), and a TATA box. Primer extension analysis revealed that the predominant transcription initiation sites for CYP19A1 and A2 transcripts were 28 and 91 bp upstream from the putative translation initiation codon, respectively. These analyses indicate that substantially different regulators, including a variety of environmental xenobiotics, control the expression the two CYP19 genes.
Subject(s)
5' Untranslated Regions/genetics , Aromatase/genetics , Zebrafish/genetics , Animals , Base Sequence , DNA/analysis , Molecular Sequence Data , Transcription, GeneticABSTRACT
Molecular cloning of the channel catfish FSH receptor is reported together with temporal changes in the gene expression throughout a reproductive cycle. A cDNA encoding the receptor was isolated from the testis using reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) procedures. The cDNA coded for a 662-amino acid protein that was most identical (51%-59%) to salmon gonadotropin receptor I and the FSH receptors of higher vertebrates, and less identical to LH receptors and thyrotropin receptors (45%-49% and 46%-47%, respectively). In addition, PCR analysis of the genomic DNA showed the absence of the LH receptor-specific intron. Expression of the channel catfish FSH receptor gene was highly restricted to the testis and ovary, except for a low-level expression in the spleen. Transfected COS cells expressed an active recombinant receptor as determined by the ligand-specific activation of a cAMP-responsive reporter gene (luciferase). The recombinant receptor was activated by human FSH and, to a small extent, hCG. Seasonal changes in the ovarian expression of the FSH receptor gene, examined by measuring the transcript abundance by quantitative real-time RT-PCR, showed a rise around the time of onset of ovarian recrudescence and a decrease prior to spawning. This pattern of seasonal expression of FSH receptor differs significantly from that of the LH receptor, which we reported recently. The differential expression of the two gonadotropin receptor genes, in addition to the differential secretion of the gonadotropic hormones, seem to be critical for the regulation of steroidogenesis and other gonadal physiological processes.
Subject(s)
Cloning, Molecular , DNA, Complementary/genetics , Gene Expression , Ictaluridae/genetics , Receptors, FSH/genetics , Seasons , Amino Acid Sequence , Animals , Base Sequence , COS Cells , Molecular Sequence Data , Receptors, FSH/chemistry , Receptors, LH/chemistry , Receptors, Thyrotropin/chemistry , Reverse Transcriptase Polymerase Chain Reaction , Sequence Homology , TransfectionABSTRACT
There is little known about the molecular biology of piscine gonadotropin receptors, and information about gene expression during reproductive development is particularly lacking. We have cloned the LH receptor (LHR) in the channel catfish (cc), and examined its gene expression throughout a reproductive cycle. A cDNA encoding the receptor was isolated from the testis using reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends procedures. It encoded a 696-amino acid protein that showed the greatest homology (46-50% identity) with the known LHRs and lesser similarity with FSH receptors and thyroid-stimulating hormone receptors (44-47% and 42-44% identity, respectively). In addition, two characteristics unique to the LHRs were conserved in the cloned receptor and the encoding gene: presence of an intron corresponding to intron 10 in mammals and turkey and occurrence of a double cysteine residue in the cytoplasmic tail for potential palmitoylation. The ccLHR gene was well expressed in the gonads and kidney and merely detectable in the gills, muscle, and spleen. The isolated cDNA encoded an active ccLHR protein, as the recombinant receptor expressed in COS7 cells activated a cAMP response element-driven reporter gene (luciferase) upon exposure to hCG in a dose-dependent manner. Seasonal changes in the ovarian expression of the ccLHR gene, as examined by measuring the transcript abundance by quantitative real-time RT-PCR, remained rather low during most of the reproductive cycle but was acutely induced around the time of spawning. This pattern of expression correlates well with the reported expression of its ligand (LH) in fishes and concurs with the notion that LH is a key regulator of the periovulatory maturational events.
Subject(s)
Follicular Phase/genetics , Gene Expression Regulation , Ictaluridae/physiology , Receptors, LH/genetics , Amino Acid Sequence , Animals , Base Sequence , COS Cells , DNA, Complementary/genetics , Female , Humans , Ictaluridae/genetics , Introns , Male , Molecular Sequence Data , Organ Specificity , Ovary/chemistry , Ovary/physiology , Phylogeny , RNA/chemistry , RNA/isolation & purification , Random Amplified Polymorphic DNA Technique , Reverse Transcriptase Polymerase Chain Reaction , Seasons , Sequence Homology, Amino Acid , Testis/chemistry , Testis/physiologyABSTRACT
17beta-hydroxysteroid dehydrogenase-I (17beta-HSD-I) is a key steroidogenic enzyme for estradiol-17beta (E(2)) production. cDNAs encoding 17beta-HSD-I were cloned for the first time in lower vertebrates from the ovary of a teleost, the Japanese eel. The deduced amino acid sequence from these cDNAs was approximately 50% identical to mammalian 17beta-HSD-Is. 17beta-HSD-I mRNA was not detected in previtellogenic ovaries by Northern blotting. However, transcript abundance increased in early vitellogenic ovaries obtained from fish artificially matured by gonadotropic treatment, but thereafter did not appear to change further. Recombinant 17beta-HSD-I expressed in human kidney 293 cells selectively converted estrone to E(2), but androstenedione, testosterone, or E(2) were not converted to any other steroids. Although it is widely accepted that E(2) is produced from testosterone in other species of teleosts, the substrate specificity of eel 17beta-HSD-I suggests that a steroidogenic pathway for production of E(2) from androstenedione via estrone exists in the Japanese eel ovary.
Subject(s)
17-Hydroxysteroid Dehydrogenases/genetics , Eels/genetics , Ovary/enzymology , 17-Hydroxysteroid Dehydrogenases/chemistry , 17-Hydroxysteroid Dehydrogenases/metabolism , Amino Acid Sequence , Animals , Cell Line , Chickens , Cloning, Molecular , DNA, Complementary , Female , Humans , Japan , Male , Mammals , Molecular Sequence Data , Organ Specificity , RNA, Messenger/genetics , Rats , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Sequence Alignment , Sequence Homology, Amino Acid , Substrate Specificity , Testis/enzymology , Transcription, Genetic , TransfectionABSTRACT
In vertebrates, the growth and maturation of the ovarian follicle is dependent on the appropriate dynamics of sex steroid secretion, which is dictated by gene expression of the steroidogenic enzymes. The molecular aspects of steroid regulation are poorly understood in fishes, so as a first step we determined the pattern of expression of four key steroidogenic genes throughout the ovarian cycle in an annually spawning teleost, the channel catfish (Ictalurus punctatus). The abundance of transcripts encoding 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) and cholesterol side chain cleavage (P450(scc)), 17 alpha-hydroxylase/lyase (P450(c17)), and aromatase (P450(arom)) were determined by rtqRT-PCR or ribonuclease protection assay and correlated to ovarian growth and plasma titers of estradiol (E(2)) and testosterone (T) in two populations of catfish. Elevations in transcript abundance for P450(c17), P450(scc), and P450(arom) were observed at the onset of ovarian recrudescence and during early vitellogenic growth of the oocytes; however, all three decreased precipitously with the completion of vitellogenesis. Changes in the expression of these genes strongly suggest a direct correlation to E(2) and T titers. Alternatively 3 beta-HSD transcript abundance was relatively stable throughout the year. This study suggests that the genes encoding the three steroidogenic cytochrome P450s have a similar regulatory mechanism.
Subject(s)
Ictaluridae/metabolism , Ovary/enzymology , Reproduction/physiology , Steroids/biosynthesis , Animals , Estradiol/metabolism , Female , Nuclease Protection Assays , Ovary/growth & development , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , RNA, Messenger/isolation & purification , Reverse Transcriptase Polymerase Chain Reaction , Seasons , Testosterone/metabolismABSTRACT
Cytochrome P450 aromatase (P450arom; CYP19) mediates the conversion of androgens to estrogens and its activity has been found in all vertebrates studied to date. This study describes the full-length cDNA encoding the ovarian form of P450arom and the differences in the 5'-untranslated region (5'-UTR) of the extra-gonadal P450arom transcript expressed by the Atlantic stingray (Dasyatis sabina). Elasmobranchs (cartilaginous fishes such as sharks, rays and skates) diverged from the other vertebrates more than 350 million years ago, therefore the stingray P450arom cDNA may represent an ancient form of this gene. Northern blot analysis showed that the ovarian follicle expressed transcripts of 3.1 and 1.7 kb in size which correspond to the clones isolated from a stingray ovarian follicle cDNA library. Both transcripts consisted of an identical 1.5 kb coding region and a 41 bp 5'-UTR, however the 3'-UTRs differed in the use of the most proximate and the most distal of four polyadenylation signals. COS cells transfected with the 1.7 kb cDNA had twice the aromatase activity as cells transfected with the 3.1 kb cDNA. The coding region of the cDNA predicted a 58.5 kDa protein which consisted of 511 residues. Alignment of the stingray protein indicates that the P450arom is equally identical (53-59%) to all other vertebrate forms of P450arom characterized to date, thus indicating a common ancestry. The evolutionary relationship of the stingray form of P450arom clearly predates the other forms and belongs to a unique lineage. Transcripts of P450arom were expressed in ovarian follicles (of all sizes), the testis, the pituitary, in all sections of the brain, and in the kidney. The extra-gonadal transcripts appear to encode a protein identical to the ovarian form, however, the 5'-UTR was 657 bp longer presumably due to the transcription of an untranslated 'first exon' as seen in the mammalian form of this gene.
Subject(s)
Aromatase/genetics , DNA, Complementary/genetics , Amino Acid Sequence , Animals , Aromatase/metabolism , Base Sequence , COS Cells , DNA, Complementary/analysis , Evolution, Molecular , Fishes , Molecular Sequence Data , Organ Specificity , Sequence AlignmentABSTRACT
The thyroid stimulating hormone receptor (TSHR) mediates the pituitary control of the development, growth and function of the thyroid. The expression of the gene encoding this receptor is known only in the thyroid, lymphocytes, fibroblasts, retro-orbital tissue and fat cells. We have cloned a TSHR from the gonads of a non-mammalian vertebrate, a bony fish (striped bass, Morone saxatilis) in the course of our search for gonadotropin receptors (follicle stimulating hormone receptor, FSHR and luteinizing hormone receptor, LHR). RT-PCR analysis demonstrated that the striped bass TSHR (stbTSHR) transcripts were abundant in both the thyroid and gonads and detectable in skeletal muscle, heart and brain tissues. The stbTSHR cDNA encoded a 779-amino acid glycoprotein hormone receptor with much higher homology (57-59%) to the mammalian TSH receptors than the mammalian LH receptors (47-49%) and FSH receptors (47%), and salmon and catfish gonadotropin receptors (42-45%). There was a TSHR-specific insertion in the extracellular domain as seen in mammalian receptors. Moreover, PCR analysis of genomic DNA indicated the absence of the LHR-specific intron in the striped bass TSHR gene. Recombinant stbTSHR expressed in COS1 cells activated reporter genes (luciferase) driven by either a cAMP response element or the c-fos promoter in response to bovine TSH, stbLH or hCG, but not human FSH. In situ hybridization studies revealed the presence of stbTSHR transcripts in the gametes but not in the follicular cells. This pattern of expression is unique and suggests a direct, albeit unknown, role for TSH in gamete physiology.
Subject(s)
Bass/genetics , Ovary/physiology , Receptors, Thyrotropin/genetics , Testis/physiology , Thyrotropin/metabolism , Amino Acid Sequence , Animals , Bass/physiology , COS Cells , Cattle , Cloning, Molecular , DNA, Complementary , Female , Genes, Reporter , Humans , In Situ Hybridization , Male , Molecular Sequence Data , Ovary/ultrastructure , Phylogeny , RNA, Messenger/metabolism , Receptors, Thyrotropin/chemistry , Receptors, Thyrotropin/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Sequence Alignment , Sequence Analysis, DNA , Testis/ultrastructure , Thyroid Gland/metabolismABSTRACT
As a first step in investigating the mechanism underlying the steroidogenic shift from the production of ovarian androgens (vitellogenic stage) to that of 17alpha-hydroxylated progestins (maturational stage) in Japanese eel during induced oogenesis, a cDNA encoding Japanese eel (Anguilla japonica) ovarian P450c17 (CYP17: steroid 17alpha-hydroxylase/C(17-20) lyase) was cloned and sequenced. This cDNA contained the complete coding region representing 510 amino acid residues, which showed high sequence homology to those of rainbow trout (74%) and mammals (45-55%). The protein encoded by this cDNA possessed high enzymatic activities of 17alpha-hydroxylase and C(17-20) lyase, thus quickly converting pregnenolone and progesterone to their respective delta(4) and delta(5) C19 products. P450c17 produced a single transcript of 2.4 kb in length, as assessed by Northern blot. Transcript levels of this enzyme significantly increased throughout artificially induced ovarian development. Considering this together with the previous data showing that C(17-20) lyase activity decreased from the vitellogenic to the maturational stage, whereas 17alpha-hydroxylase activity increased, the present data suggest that changes in C(17-20) lyase activity (the production of androgens) do not depend on transcriptional changes of the P450c17 gene.
Subject(s)
DNA, Complementary/biosynthesis , Eels/physiology , Ovary/enzymology , Steroid 17-alpha-Hydroxylase/biosynthesis , Amino Acid Sequence , Animals , Base Sequence , Blotting, Northern , COS Cells , Cloning, Molecular , DNA, Complementary/analysis , DNA, Complementary/genetics , Female , Gene Library , Molecular Sequence Data , Poly A/biosynthesis , Poly A/genetics , Pregnenolone/biosynthesis , Progesterone/biosynthesis , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain Reaction , Species Specificity , Steroid 17-alpha-Hydroxylase/geneticsABSTRACT
To accelerate the integration of ceramic implants with the surrounding bone and to search for a suitable carrier for bone morphogenetic protein (BMP), we studied ectopic bone induction in porous apatite-wollastonite-containing glass ceramic (A-W GC) combined with partially purified bovine BMP (bBMP) and recombinant Xenopus BMP-4/7 (rxBMP-4/7). Porous A-W GC rods [4 mm in diameter, 5 mm in height, 70% porosity, 200 microns mean pore size, 17.54 +/- 3.82 MPa (mean +/- SD) compressive strength] were used. An apatite coating formed on the surface of porous A-W GC that had been immersed in simulated body fluid at 36.5 degrees C for 7 days. In experiment 1, porous A-W GC rods were combined with either bBMP, collagen, or with both bBMP and collagen. The rods were implanted into subcutaneous pouches in rats and were harvested 4 weeks after implantation. Low-energy radiographic, scanning electron microscopic (SEM), and histological examinations showed ectopic bone formation and within the rods only in the porous A-W GC combined with the bBMP and collagen group. Quantitative analysis also revealed that this group alone showed a significant increase in bone formation. In experiment 2, porous A-W GC rods were combined with rxBMP and collagen, implanted into rats, and harvested as described above. SEM and histological examination showed ectopic bone formation around and within the rods. Because of its relatively high mechanical strength, ease of handling, and good osteoinductivity, porous A-W GC combined with BMP and collagen may be clinically useful in patients with large cancellous bone defects or craniomaxillofacial lesions.
Subject(s)
Apatites , Bone Development , Bone Morphogenetic Proteins , Calcium Compounds , Ceramics/chemistry , Glass/chemistry , Silicates , Animals , Cattle , Collagen , Microscopy, Electron, Scanning , Rats , Recombinant Proteins , XenopusABSTRACT
The role of nitric oxide (NO) in the pathogenesis of influenza virus-induced pneumonia in mice was investigated. Experimental influenza virus pneumonia was produced with influenza virus A/Kumamoto/Y5/67(H2N2). Both the enzyme activity of NO synthase (NOS) and mRNA expression of the inducible NOS were greatly increased in the mouse lungs; increases were mediated by interferon gamma. Excessive production of NO in the virus-infected lung was studied further by using electron spin resonance (ESR) spectroscopy. In vivo spin trapping with dithiocarbamate-iron complexes indicated that a significant amount of NO was generated in the virus-infected lung. Furthermore, an NO-hemoglobin ESR signal appeared in the virus-infected lung, and formation of NO-hemoglobin was significantly increased by treatment with superoxide dismutase and was inhibited by N(omega)-monomethyl-L-arginine (L-NMMA) administration. Immunohistochemistry with a specific anti-nitrotyrosine antibody showed intense staining of alveolar phagocytic cells such as macrophages and neutrophils and of intraalveolar exudate in the virus-infected lung. These results strongly suggest formation of peroxynitrite in the lung through the reaction of NO with O2-, which is generated by alveolar phagocytic cells and xanthine oxidase. In addition, administration of L-NMMA resulted in significant improvement in the survival rate of virus-infected mice without appreciable suppression of their antiviral defenses. On the basis of these data, we conclude that NO together with O2- which forms more reactive peroxynitrite may be the most important pathogenic factors in influenza virus-induced pneumonia in mice.
Subject(s)
Nitric Oxide/metabolism , Pneumonia, Viral/physiopathology , Reactive Oxygen Species/metabolism , Animals , Arginine/analogs & derivatives , Arginine/pharmacology , Electron Spin Resonance Spectroscopy , Enzyme Induction , Enzyme Inhibitors/pharmacology , Free Radicals , Influenza A virus/pathogenicity , Interferon-gamma/physiology , Lung/metabolism , Male , Mice , Nitric Oxide Synthase/antagonists & inhibitors , Nitric Oxide Synthase/biosynthesis , Superoxides/metabolism , omega-N-MethylarginineABSTRACT
To elucidate the mechanism of bacterial exoprotease in promotion of the intravascular dissemination of Pseudomonas aeruginosa, we examined the possible involvement of bradykinin (whose generation is induced by pseudomonal proteases in septic foci) in the invasion by bacteria, and in access of bacterial toxins to systemic blood circulation. P. aeruginosa 621 (PA 621), which produces very little protease, was injected intraperitoneally into mice together with pseudomonal exoproteases (elastase/alkaline protease). Dissemination of bacteria from the peritoneal septic foci to the blood was assessed by counting viable bacteria in the blood and spleen by use of the colony-forming assay. The results showed that pseudomonal proteases markedly enhanced (10- to 100-fold) intravascular dissemination of bacteria in mice. This enhancement was induced not only by pseudomonal proteases but also by bradykinin. More importantly, the increased spread of PA 621 induced by pseudomonal protease and bradykinin was significantly augmented by the addition of kininase inhibitors, indicating the direct involvement of bradykinin in bacterial dissemination. Similarly, bradykinin caused effective dissemination of pseudomonal toxins such as endotoxin (lipopolysaccharide) and exotoxin A when the toxins were injected into the peritoneal cavity with bradykinin. Furthermore, the lethality of the infection with PA 621 was strongly enhanced by pseudomonal proteases given i.p. simultaneously with PA 621. On the basis of these results, it is strongly suggested that pseudomonal proteases as well as bradykinin generated in infectious foci are involved in facilitation of bacterial dissemination in vivo.
Subject(s)
Bacterial Proteins , Bradykinin/metabolism , Metalloendopeptidases/pharmacology , Pseudomonas Infections/metabolism , Pseudomonas aeruginosa/drug effects , Sepsis/metabolism , Serine Endopeptidases/pharmacology , Animals , Male , Mice , Pseudomonas Infections/microbiology , Pseudomonas aeruginosa/growth & development , Pseudomonas aeruginosa/isolation & purification , Sepsis/microbiologyABSTRACT
Demineralized bone matrix and bone morphogenetic protein have been used clinically to accelerate bone regeneration. However, the best method of sterilization has been the subject of controversy. Some investigators have used ethylene oxide, but others have reported that doses adequate for sterilization destroyed the osteoinductivity of demineralized bone matrix and that gamma irradiation was less harmful in this respect. We used partially purified bone morphogenetic protein and type-I collagen to investigate the effects of sterilization by ethylene oxide and gamma irradiation on the activity of bone morphogenetic protein. Osteoinductivity was reduced considerably after sterilization by gamma irradiation at 2.5 Mrad and by ethylene oxide at 37 degrees C for 4 hours and at 55 degrees C for 1 hour; however, the reduction induced by ethylene oxide at 29 degrees C for 5 hours was about half of the control values. This study showed that ethylene oxide at 29 degrees C for 5 hours can be used clinically for sterilization of bone morphogenetic protein. We also investigated the effect of gamma irradiation on bone morphogenetic protein and the collagen carrier separately and found that collagen was far more labile than bone morphogenetic protein.
Subject(s)
Proteins/physiology , Sterilization/methods , Animals , Bone Development/drug effects , Bone Development/radiation effects , Bone Morphogenetic Proteins , Bone Transplantation , Cattle , Collagen/drug effects , Collagen/physiology , Collagen/radiation effects , Ethylene Oxide , Gamma Rays , Male , Proteins/drug effects , Proteins/radiation effects , Rats , Rats, Sprague-DawleyABSTRACT
The antimicrobial action of .NO against Cryptococcus neoformans was investigated by using imidazolineoxyl N-oxide, which we recently reported removes .NO via oxidation (T. Akaike, M. Yoshida, Y. Miyamoto, K. Sato, M. Kohno, K. Sasamoto, K. Miyazaki, S. Ueda, and H. Maeda, Biochemistry 32:827-832, 1993). No appreciable fungicidal activity was observed in neutral .NO solutions. Imidazolineoxyl N-oxide induced or enhanced fungicidal action in neutral or acidic .NO solutions, respectively. Our results provide convincing evidence that .NO is not a microbicidal molecular species.
Subject(s)
Anti-Infective Agents/pharmacology , Cyclic N-Oxides/pharmacology , Imidazoles/pharmacology , Nitric Oxide/pharmacology , Cryptococcus neoformans/drug effects , Nitric Oxide/metabolismABSTRACT
A study was conducted to investigate the influence of systemic inflammation on heterotopic bone formation induced by bone morphogenetic protein (BMP). Five-milligram pellets of BMP were implanted in mice with type II collagen-induced arthritis. Intraperitoneal injections of interleukin-1 (IL-1) were also administered to a group of mice without collagen-induced arthritis. The amount of BMP-induced heterotopic bone formation was evaluated by soft X-ray radiography, histology, and assay of calcium content. BMP-induced heterotopic bone formation was markedly enhanced in mice with collagen-induced arthritis, and also in IL-1-treated mice. These findings suggest that bone formation is enhanced in mice with collagen-induced arthritis, and that IL-1 may be responsible.