Anti-Melanogenesis Activity of Crocodile (Crocodylus siamensis) White Blood Cell Extract on Ultraviolet B-Irradiated Melanocytes.

Ultraviolet (UV) radiation generates a range of biological effects in the skin, which includes premature skin aging, hyperpigmentation, and cancer. Therefore, the development of new effective agents for UV-related skin damage remains a challenge in the pharmaceutical industry. This study aims to test the inhibitory effect of crocodile white blood cell (cWBC) extract, a rich source of bioactive peptides, on ultraviolet B (UVB)-induced melanocyte pigmentation. The results showed that cWBC (6.25-400 µg/mL) could inhibit tyrosinase without adduct formation by 12.97 ± 4.20% on average. cWBC pretreatment (25-100 µg/mL) had no cytotoxicity and reduced intracellular melanin to 111.17 ± 5.20% compared with 124.87 ± 7.43 for UVB condition. The protective role of cWBC pretreatment against UVB was exhibited by the promotion of cell proliferation and the prevention of UVB-induced morphological change as observed from F actin staining. The decrease of microphthalmia-associated transcription factor expression levels after cWBC pretreatment might be a mechanism by which cWBC suppresses UVB-induced pigmentation. These results suggest that cWBC could be beneficial for the prevention of UVB-induced skin pigmentation.

Ghrelin and ghrelin receptor (GHSR) in Chinese alligator, alligator sinensis: Molecular characterization, tissue distribution and mRNA expression changes during the active and hibernating periods.

The Chinese alligator (Alligator sinensis) is a freshwater crocodilian endemic to China. So far, the endocrine regulation of feeding and growth in Chinese alligator is poorly understood. In this study, the molecular structure and tissue expression profiles of ghrelin and its receptor GHSR in the Chinese alligator were characterized for the first time. The full-length cDNA of ghrelin was 1770 bp, including a 37 bp 5 '-UTR (untranslated region), a 435 bp ORF (open reading frame) and a 1298 bp 3 '-UTR. The ORF encodes a ghrelin precursor, which consists of 145 amino acid residues, including a signal peptide with 52 amino acid residues at the N-terminus, a mature peptide with 28 amino acid residues, and a possibly obestain at the C-terminus. The full-length cDNA of GHSR was 3961 bp, including a 5'-UTR of 375-bp, an ORF of 1059-bp and a 3' -UTR of 2527-bp. The ORF encodes a protein of 352 amino acid residues containing seven transmembrane domains, with multiple N glycosylation modification sites and conserved cysteine residue sites. The active core "GSSF" of Chinese alligator ghrelin was identical to that of mammals and birds, and the ghrelin binding site of GHSR was similar to that of mammals. The amino acid sequences of both ghrelin and GHSR share high identity with American alligator (Alligator mississippiensis) and birds. Ghrelin was highly expressed in cerebrum, mesencephalon, hypothalamus and multiple peripheral tissues, including lung, stomach and intestine, suggesting that it could play functions in paracrine and/or autocrine manners in addition to endocrine manner. GHSR expression level was higher in hypothalamus, epencephalon and medulla oblongata, and moderate in multiple peripheral tissues including lung, kindey, stomach and oviduct, implicating that ghrelin/GHSR system may participate in the regulation of energy balance, food intake, water and mineral balance, gastrointestinal motility, gastric acid secretion and reproduction. During hibernation, the expression of ghrelin and GHSR in the brain was significantly increased, while ghrelin was significantly decreased in heart, liver, lung, stomach, pancreas and ovary, and GHSR was significantly decreased in heart, liver, spleen, lung, kindey, stomach, ovary and oviduct. These temporal changes in ghrelin and GHSR expression could facilitate the physiological adaption to the hibernation of Chinese alligator. Our study could provide basic data for further studies on the regulation of feeding, physiological metabolism and reproduction of Chinese alligator, which could also be useful for the improvement of artificial breeding of this endangered species.

Stress responses in captive Crocodylus moreletii associated with metal exposure.

Environmental pollution by metals has repercussions on wildlife health. It is known that some metals can have an influence on the neuroendocrine stress response, and at the same time, metals have pro-oxidant effects that can overwhelm the antioxidant system and cause oxidative stress. This study evaluates the association of metals with neuroendocrine stress activity and biomarkers of oxidative stress in 42 captive female Morelet's crocodiles (Crocodylus moreletii). We measured five metals of ecotoxicological importance (Hg, Cd, Pb, Cu and Zn), and three biomarkers of the oxidative stress response in the liver: glutathione (GSH) and glutathione disulfide (GSSG) as markers for antioxidant system and thiobarbituric acid reactive substances (TBARS) for oxidative damage. We also measured one biomarker of the neuroendocrine response to stress: corticosterone (B) in blood plasma. The mean ± SD concentrations of metals in the liver expressed in µg/g (dw) were: Cd: 0.004 ± 0.003, Hg: 0.014 ± 0.019, Cu: 0.017 ± 0.013, Zn: 0.043 ± 0.035, Pb: 0.16 ± 0.256. The mean ± SD of GSH was 0.42 ± 0.35 nmol/mg protein, the mean ± SD of GSSG was 0.24 ± 0.20 nmol/mg protein, the mean ± SD concentrations of TBARS were 0.36 ± 0.21 nmol/mg protein, and the mean ± SD of B was 393.57 ± 405.14 pg/mL. Hg presented a significant negative relationship with corticosterone. Cd had a negative relationship with both GSH and GSSG; meanwhile, Zn showed a negative relationship with TBARS levels, could be a protective element against hepatic oxidative damage. Finally, B had negative relationship with oxidative damage. The connection found between Hg and the neuroendocrine stress response, as well as the correlations of Cd and Zn with oxidative damage and antioxidant activity should be studied further, given their toxicological importance and implications for the conservation of C. moreletii and other crocodilians.

Molecular cloning, immunological characterization, and expression analysis of gonadotropin-releasing hormone (GnRH) in the brain of the Chinese alligator during different stages of reproductive cycle.

The neurohormone gonadotropin-releasing hormone (GnRH) plays an essential role in the control of reproductive functions in vertebrates. However, the full-length complementary DNA (cDNA) encoding the GnRHs precursor and it role in the reproductive cycles regulating has not been illustrated in crocodilian species. In the present study, full-length cDNAs encoding GnRH1 forms, its predominant localization within brain and peripheral tissues, and GnRH1 peptide concentrations in the hypothalamus and pituitary in relation to seasonal gonadal development of Chinese alligator were investigated. The cDNA of GnRH1 is consisted of 282 bp open reading frame encoding 93 amino acids. The deduced amino acid sequence of alligator GnRH1 contains several conserved regions and shows a closer genetic relationship to the avian species than to other reptile species. The GnRH1 immunopositive cells were not only detected widely in cerebrum, diencephalon, medulla oblongata but also observed in peripheral tissues, these widespread distribution characteristics indicated that GnRH1 possibly possess the multi-functionality in Chinese Alligator. GnRH1 peptide concentration within hypothalamus were observed be the highest in RP group (P < 0.05), in association with an peak value in GSI and emerging of late vitellogenic follicles in the ovary. Taken together, our results suggested that GnRH1 was predominantly involved in the vitellogenesis process of seasonal gonadal development of Chinese Alligator.

Salt-water acclimation of the estuarine crocodile Crocodylus porosus involves enhanced ion transport properties of the urodaeum and rectum.

Estuarine crocodiles, Crocodylus porosus, inhabit freshwater, estuarine and marine environments. Despite being known to undertake extensive movements throughout and between hypo-osmotic and hyperosmotic environments, little is known about the role of the cloaca in coping with changes in salinity. We report here that, in addition to the well-documented functional plasticity of the lingual salt glands, the middle of the three cloacal segments (i.e. the urodaeum) responds to increased ambient salinity to enhance solute-coupled water absorption. This post-renal modification of urine serves to conserve water when exposed to hyperosmotic environments and, in conjunction with lingual salt gland secretions, enables C. porosus to maintain salt and water balance and thereby thrive in hyperosmotic environments. Isolated epithelia from the urodaeum of 70% seawater-acclimated C. porosus had a strongly enhanced short-circuit current (an indicator of active ion transport) compared with freshwater-acclimated crocodiles. This enhanced active ion absorption was driven by increased Na+/K+-ATPase activity, and possibly enhanced proton pump activity, and was facilitated by the apical epithelial Na+ channel (ENaC) and/or the apical Na+/H+ exchanger (NHE2), both of which are expressed in the urodaeum. NHE3 was expressed at very low levels in the urodaeum and probably does not contribute to solute-coupled water absorption in this cloacal segment. As C. porosus does not appear to drink water of salinities above 18 ppt, observations of elevated short-circuit current in the rectum as well as a trend for increased NHE2 expression in the oesophagus, the anterior intestine and the rectum suggest that dietary salt intake may stimulate salt and possibly water absorption by the gastrointestinal tract of C. porosus living in hyperosmotic environments.

Molecular cloning of androgen receptor and gene expression of sex steroid hormone receptors in the brain of newborn Chinese alligator (Alligator sinensis).

Sex steroid hormones play an important role in mediating physiological responses and developmental processes through their receptors across all vertebrates. Chinese alligator (Alligator sinensis) is a critically endangered reptile species unique to China. In this study, we have cloned one of the sex steroid hormone receptor genes, androgen receptor (AR) from the brain of Chinese alligator for the first time. The full-length AR cDNA is 2717 bp in length with an open reading frame (ORF) encoding 722 amino acids. Amino acid alignment analyses indicated that the ARs exhibit highly conserved functional domains. Especially, the P-box and D-box, which are essential to ensure that receptor binding to the androgen response elements, are completely conserved in selected species. Using the quantitative real-time PCR (qPCR), the spatial expression of four receptor mRNAs in all newborn brain tissues and temporal expression of them in the cerebrum during the embryonic development in Chinese alligators were investigated. The results of qPCR showed ubiquitous expression of the four receptor mRNAs in all newborn brain tissues examined and significant changes in the expression levels of these receptor mRNAs in the embryonic development. These results suggest that sex steroid hormones might play an important role in the regulation of complex neuroendocrine activities in newborn Chinese alligator. Furthermore, these data provide an important foundation for further studies on endocrinology and molecular biology of non-mammalian sex steroid hormone receptors.

Divergent responsiveness of two isoforms of the estrogen receptor to mixtures of contaminants of emerging concern in four vertebrates.

Contaminants of emerging concern (CECs) are ubiquitous in aquatic environments with well-established endocrine-disrupting effects. A data matrix of 559 water samples was queried to identify two commonly occurring CECs mixtures in Great Lakes tributaries. One mixture consisted of eight agricultural CECs (AG), while another contained 11 urban CECs (UB). The known estrogenic compounds bisphenol A, estrone and nonylphenol were present in both mixtures. According to the EPA Tox21 in ToxCast database, AG and UB mixture at an environmentally relevant concentration were estimated to account for 6.5% and 3.4% estrogenicity of the model endocrine disruptor estradiol-17ß, respectively. Two isoforms of the estrogen receptor (Esr1 and -2, former Erα and Erß) cloned from fathead minnow, bluegill sunfish, American alligator and human, responded differently to AG and UB mixtures. Human and bluegill Esr1 were the most sensitive to AG and UB mixtures, respectively. Fathead minnow Esr1 and Esr2b were the least sensitive to 10× AG and UB in estrogen dose equivalents, respectively. Even at environmentally documented concentrations, UB significantly activated bluegill Esr1. Moreover, 100× concentrated UB hyperstimulated fathead minnow Esr1 beyond the maximum induction of estradiol-17ß. These results indicate that efficacious receptors and species differ in their response to CEC mixtures. Furthermore, estrogenicity may be present in some CECs not previously considered estrogenic, or, alternatively, estrogenicity of a mixture may be enhanced through chemical interactions. Our study highlights the need for further studies of CECs utilizing a variety of receptors cloned from diverse species.

Accumulation of organochlorine pesticides in fat tissue of wild Nile crocodiles (Crocodylus niloticus) from iSimangaliso Wetland Park, South Africa.

Nile crocodiles (Crocodylus niloticus) are important apex predators in many tropical and subtropical aquatic habitats throughout much of sub-Saharan Africa. In South Africa, large crocodile populations inhabit lakes and wetlands that are impacted by organochlorine pesticides (OCPs). Despite the continued use of these compounds and their potential adverse effects on key wildlife populations in southern Africa, limited ecotoxicoloigcal data exist. In this study, we examined the accumulation of OCPs in fat tissues of live, wild Nile crocodiles from iSimangaliso Wetland Park, a region of significant biological importance. All samples (n = 15) contained multiple contaminants in highly elevated concentrations, with total residue burdens varying between 3600 and 8000 ng g-1 ww. DDT and its metabolites were the dominant compounds detected in most samples, with ∑DDT concentrations ranging between 520 and 3100 ng g-1 ww. Elevated levels of other OCPs were also detected, including lindane (67-410 ng g-1 ww), aldrin (150-620 ng g-1 ww) and heptachlor (170-860 ng g-1 ww). Our findings show that crocodiles are exposed to OCPs throughout their range within iSimangaliso Wetland Park and contain some of the highest concentrations ever recorded in crocodilian tissue. Results indicate the need for a greater understanding of the impacts of OCP exposure and toxicological responses in crocodiles from iSimangaliso, and in Nile crocodile populations in general. The novel surgical technique described in this study provides an effective method for assessing relationships between contaminant body burdens and their potential reproductive and developmental consequences in crocodilians.

Feminizing effects of exposure to Corexit-enhanced water-accommodated fraction of crude oil in vitro on sex determination in Alligator mississippiensis.

Deepwater Horizon spilled over 200 million gallons of oil into the waters of the Gulf of Mexico in 2010. In an effort to contain the spill, chemical dispersants were applied to minimize the amount of oil reaching coastal shorelines. However, the biological impacts of chemically-dispersed oil are not well characterized, and there is a particular lack of knowledge concerning sublethal long-term effects of exposure. This study examined potential estrogenic effects of CWAF, Corexit 9500-enhanced water-accommodated fraction of oil, by examining its effect on estrogen receptors and sex determination in the American alligator, Alligator mississippiensis. The alligator exhibits temperature-dependent sex determination which is modulated by estrogen signals, and exposure to 17ß-estradiol (E2) and estrogenic compounds in ovo during the thermosensitive period of embryonic development can induce ovarian development at a male-producing temperature (MPT). CWAF induced transactivation up to 50% of the maximum induction by E2 via alligator estrogen receptors in vitro. To determine potential endocrine-disrupting effects of exposure directly on the gonad, gonad-adrenal-mesonephric (GAM) organ complexes were isolated from embryos one day prior to the thermosensitive period and exposed to E2, CWAF, or medium alone in vitro for 8-16 days at MPT. Both CWAF and E2 exposure induced a significant increase in female ratios. CWAF exposure suppressed GAM mRNA abundances of anti-Müllerian hormone (AMH), sex determining region Y-box 9, and aromatase, whereas E2 exposure suppressed AMH and increased Forkhead box protein L2 mRNA abundances in GAM. These results indicate that the observed endocrine-disrupting effects of CWAF are not solely estrogenically mediated, and further investigations are required.

Crocodiles and alligators: Antiamoebic and antitumor compounds of crocodiles.

Crocodiles exist in unsanitary environments, feed on rotten meat, are often exposed to heavy metals such as arsenic, cadmium, cobalt, chromium, mercury, nickel, lead, selenium, tolerate high levels of radiation, and are amid the very few species to survive the catastrophic Cretaceous-Tertiary extinction event, nonetheless they can live for up to a 100 years. Moreover, as they live in unhygienic conditions, they regularly come across pathogens. Logically, we postulate that crocodiles possess mechanisms to defend themselves from noxious agents as well as protecting themselves from pathogens. To test this hypothesis, various organ lysates and serum of Crocodylus palustris were prepared. Amoebicidal assays were performed using Acanthamoeba castellanii belonging to the T4 genotype. Cytotoxicity assays were performed using Prostate cancer cells culture by measuring lactate dehydrogenase release as a marker for cell death. Growth inhibition assays were performed to determine the growth inhibitory effects of various organ lysates. Serum and heart lysates of Crocodylus palustris exhibited powerful anti-tumor activity exhibiting more than 70% Prostate cancer cell death (P < 0.05). Additionally, lysates from gall bladder and bile also showed significant host cell cytotoxicity, however intestine, lungs and brain showed partial cytotoxicity. Both sera and heart lysates of Crocodylus palustris abolished Prostate cells growth. Moreover, serum completely abolished A. castellanii viability. For the first time, these findings showed that the organ lysates of Crocodylus palustris exhibit potent anti-amoebic and anti-tumor activity. The discovery of antimicrobial and antitumor activity in crocodile will stimulate research in finding therapeutic molecules from unusual sources, and has potential for the development of novel antitumor/antimicrobial compound(s) that may also overcome drug resistance. Nevertheless, rigorous research in the next few years will be necessary to realize these expectations.

Bisphenol A disrupts the temporal pattern of histofunctional changes in the female reproductive tract of Caiman latirostris.

Recently, we have described the ontogeny of histofunctional differentiation changes in the oviduct of Caiman latirostris. The expression of estrogen receptor alpha and progesterone receptor shows that the caiman oviduct could be a target of the action of xenoestrogens such as the widely environmentally present Bisphenol A (BPA), early in life. The aims of this study were: to complement oviduct characterization by establishing the ontogenetic changes in androgen receptor (AR) expression and assessing the effects of early postnatal exposure to 17-ß-estradiol (E2) or BPA on the histofunctional features of the oviduct. AR was expressed in all the stages studied. The spatial pattern of AR immunostaining changed from neonatal to juvenile caimans. In the luminal epithelium, changes were at the subcellular level, from cytoplasmic to nuclear. In the subepithelium, although both cytoplasmic and nuclear AR expression was observed, changes were mainly at tissue level, from the subepithelial compartment to the outer muscular layer. The oviduct was highly sensitive to E2 and BPA at the early postnatal developmental stage. E2- and BPA-exposed caimans showed increased luminal epithelial height and higher proliferative activity. Changes in histomorphological features (measured by a scoring system), steroid hormone receptors, collagen remodeling and muscle-associated proteins suggest a precocious oviduct histofunctional differentiation in E2- and BPA-exposed caimans. The modification of the temporal pattern of oviductal biomarkers suggests that organizational changes could impair C. latirostris reproductive health later in life. The alterations in the caiman female reproductive tract exposed to BPA highlight the importance of preserving aquatic environments from plastic pollution.

A novel antibacterial peptide derived from Crocodylus siamensis haemoglobin hydrolysate induces membrane permeabilization causing iron dysregulation, oxidative stress and bacterial death.

AIMS: A novel antibacterial peptide from Crocodylus siamensis haemoglobin hydrolysate (CHH) was characterized for antimicrobial activity. METHODS AND RESULTS: CHHs were hydrolysed for 2 h (2 h-CHH), 4 h (4h-CHH), 6 h (6 h-CHH) and 8 h (8 h-CHH). The 8 h-CHH showed antibacterial activity against Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae and Pseudomonas aeruginosa at concentrations of 20, 20, 20 and 10 mg ml-1 (w/v) respectively. Fluorescent microscopy revealed that the 8 h-CHH had bactericidal activity against E. coli and P. aeruginosa. ß-galactosidase assay supported by RT-qPCR demonstrated that the 8 h-CHH resulted in differential expression of genes involved in iron homeostasis (ftnA and bfd) and oxidative stress (sodA, soxR and oxyR). Siderophore assay indicated that the 8 h-CHH also impaired siderophore production with diminished expression of pvdF. This pattern of gene expression suggests that the 8 h-CHH triggers the release of free ferric ions in the cytoplasm. However, decreased expression of genes associated with the SOS response (recA and lexA) in combination with neutral comet revealed that no DNA damage was caused by 8 h-CHH. Membrane permeabilization assay indicated that 8 h-CHH caused membrane leakage thought to mediate the antibacterial and iron-stress responses observed, due to loss of regulated iron transport. The novel active peptide from 8 h-CHH was determined as QAIIHNEKVQAHGKKVL (QL17), with 41% hydrophobicity and +2 net charge. CONCLUSIONS: The QAIIHNEKVQAHGKKVL fragment of C. siamensis haemoglobin is antibacterial via a mechanism that likely relies on iron dysregulation and oxidative stress which results in bacterial death. SIGNIFICANCE AND IMPACT OF THE STUDY: We have described for the first time, a novel peptide derived from C. siamensis haemoglobin hydrolysate that has the potential to be developed as a novel antimicrobial peptide.

Purification, characterization, cloning and structural analysis of Crocodylus siamensis ovotransferrin for insight into functions of iron binding and autocleavage.

Ovotransferrin (OTf), the major protein constituent of egg white, is of great interest due to its pivotal role in biological iron transport and storage processes and its spontaneous autocleavage into peptidic fragments with alternative biological properties, such as antibacterial and antioxidant activities. However, despite being well-investigated in avian, a detailed elucidation of the structure-function relationship of ovotransferrins in the closely related order of Crocodilia has not been reported to date. In this study, electron paramagnetic resonance (EPR) confirmed the presence of two spectroscopically distinct ferric iron binding sites in Crocodylus siamensis OTf (cOTf), but implied a five-fold lower quantity of bound iron than in hen OTf (hOTf). In addition, quantitative estimation of free sulfhydryl groups revealed slight differences to hOTf. To gain a better structural understanding of cOTf, we found a cOTf gene consisting of an open reading frame of 2040bp and encoding a protein of 679 amino acids. In silico prediction of the three-dimensional structure of cOTf and comparison with hOTf revealed four evolutionarily conserved iron-binding sites in both N- and C-lobes, as well as the presence of only 13 of the 15 disulfide bonds in hOTf. This evolutionary loss of disulfide linkages in conjunction with the lack of hydrogen bonding from a dilysine trigger in the C-lobe are presumed to affect the iron binding and autocleavage character of cOTf. As a result, cOTf may be capable of exerting a more diverse array of functions compared to its avian counterparts; for instance, ion buffering, antioxidant and antimicrobial activities.

Detection of a synthetic sex steroid in the American crocodile (Crocodylus acutus): Evidence for a novel environmental androgen.

Endocrine-disrupting contaminants (EDC's) are well known to alter sexual differentiation among vertebrates via estrogenic effects during development, particularly in organisms characterized by temperature-dependent sex determination. However, substances producing androgenic effects typically lack potency when tested in laboratory settings and are virtually unstudied in field settings. Here, we assay levels of a synthetic androgen, 17α-methyltestosterone (MT), in a heavily male-biased population of American crocodiles in the Tempisque River Basin of Costa Rica based on the recent hypothesis that this chemical is an EDC in developing crocodilian embryos. The presence of MT was documented in all field-collected samples of egg yolk and in plasma of all age classes in among population of crocodiles. Hatchlings exhibited higher plasma MT concentrations (102.1 ± 82.8 ng/mL) than juveniles (33.8 ± 51.5) and adults (25.9 ± 20.8 ng/mL). Among populations, crocodiles captured in the Tempisque River (62.9 ± 73.7 ng/mL) were higher in MT concentration than those from Tarcoles (13.3 ± 11.4 ng/mL) and negative controls (0.001 ± 0.0002 ng/mL). A mechanism for the bio-transport of MT and its subsequent effects is proposed.

Crocodile choline from Crocodylus siamensis induces apoptosis of human gastric cancer.

Crocodile choline, an active compound isolated from Crocodylus siamensis, was found to exert potent anti-cancer activities against human gastric cancer cells in vitro and in vivo. Our study revealed that crocodile choline led to cell cycle arrest at the G2/M phase through attenuating the expressions of cyclins, Cyclin B1, and CDK-1. Furthermore, crocodile choline accelerated apoptosis through the mitochondrial apoptotic pathway with the decrease in mitochondrial membrane potential, the increase in reactive oxygen species production and Bax/Bcl-2 ratio, and the activation of caspase-3 along with the release of cytochrome c. In addition, this study, for the first time, shows that Notch pathway is remarkably deregulated by crocodile choline. The combination of crocodile choline and Notch1 short interfering RNA led to dramatically increased cytotoxicity than observed with either agent alone. Notch1 short interfering RNA sensitized and potentiated the capability of crocodile choline to suppress the cell progression and invasion of gastric cancer. Taken together, these data suggested that crocodile choline was a potent progression inhibitor of gastric cancer cells, which was correlated with mitochondrial apoptotic pathway and Notch pathway. Combining Notch1 inhibitors with crocodile choline might represent a novel approach for gastric cancer.

Pharmacokinetics of marbofloxacin in freshwater crocodiles (Crocodylus siamensis) after intravenous and intramuscular administration.

To evaluate the fate and disposition of marbofloxacin (MBF) in freshwater crocodiles (Crocodylus siamensis), MBF was administered either intravenously (i.v.) or intramuscularly (i.m.) at a dosage of 2.0 mg/kg body weight. The concentrations of MBF in plasma were measured using high-performance liquid chromatography equipped with a fluorescence detector. The concentrations of MBF in the plasma were measurable up to 144 h after i.v. and i.m. administration. After the first 45 min, the mean pharmacokinetic profiles produced by the two administration routes were almost identical. No statistically significant differences in the pharmacokinetic parameters between the groups were observed. The half-life was long (about 2.5 days), the volume of distribution was large (about 1.44 L/kg), λz was small (0.01 h-1 ), and the clearance was slow (22.6 mL/h/kg). The absolute i.m. bioavailability (F%) was 105.36%. The dose of MBF administered in this study seems to produce appropriate PK-PD parameters that predict antibacterial success for disease caused by susceptible bacteria. More studies are warranted to evaluate the likely residues after administration of multiple doses.

Insulin-like growth factor I (IGF-I) in Chinese alligator, Alligator sinensis: Molecular characterization, tissue distribution and mRNA expression changes during the active and hibernating periods.

The Chinese alligator Alligator sinensis is an endangered species endemic to China, up to date, little is known about the regulation of its growth and development. Insulin-like growth factor I (IGF-I) plays a vital role in regulating vertebrate growth and development. In this study, the full-length cDNA of IGF-I in Chinese alligator (caIGF-I) was obtained for the first time, it contains 890-bp nucleotides encoding a 153-amino acid precursor, the mature caIGF-I consists of 70 amino acids by cleaving the signal peptide and C-terminal extension (E domain). The caIGF-I contains all the features of IGF-I peptide with B, C, A, and D domains and the six conservative cysteine residues involved in the stable tertiary structure. Multiple alignment analysis showed that the amino acid sequence of caIGF-I shares high identity with American alligator Alligator mississippiensis (100%) and birds (95-97%). Phylogenetic tree analysis of the IGF-I amino acid sequences indicated that alligators cluster into the bird branch. Real-time quantitative PCR technique showed that caIGF-I is widely expressed in all the examined tissues with the highest expression level in liver, higher in pancreas and oviduct while lower in heart, spleen, lung, kidney, stomach, intestines, ovary and muscles. During hibernation, the caIGF-I expression level decreased significantly in liver, pancreas, oviduct and kidney, while did not significantly change in heart, spleen, lung, stomach, small intestine, ovary and muscles. The mRNA expression changes during the two periods implicate that caIGF-I might play an important role in the regulation of feeding and growth in the Chinese alligator.

The long road to steady state in gas exchange: metabolic and ventilatory responses to hypercapnia and hypoxia in Cuvier's dwarf caiman.

Animals with intermittent lung ventilation and those exposed to hypoxia and hypercapnia will experience fluctuations in the bodily O2 and CO2 stores, but the magnitude and duration of these changes are not well understood amongst ectotherms. Using the changes in the respiratory exchange ratio (RER; CO2 excretion divided by O2 uptake) as a proxy for changes in bodily gas stores, we quantified time constants in response to hypoxia and hypercapnia in Cuvier's dwarf caiman. We confirm distinct and prolonged changes in RER during and after exposure to hypoxia or hypercapnia. Gas exchange transients were evaluated in reference to predictions from a two-compartment model of CO2 exchange to quantify the effects of the levels of hypoxia and hypercapnia, duration of hypercapnia (30-300 min) and body temperature (23 versus 33°C). For hypercapnia, the transients could be adequately fitted by two-phase exponential functions, and slow time constants (after 300 min hypercapnia) concurred reasonably well with modelling predictions. The slow time constants for the decays after hypercapnia were not affected by the level of hypercapnia, but they increased (especially at 23°C) with exposure time, possibly indicating a temporal and slow recruitment of tissues for CO2 storage. In contrast to modelling predictions, elevated body temperature did not reduce the time constants, probably reflecting similar ventilation rates in transients at 23 and 33°C. Our study reveals that attainment of steady state for gas exchange requires considerable time and this has important implications for designing experimental protocols when studying ventilatory control and conducting respirometry.

Molecular cloning, characterization, tissue distribution and mRNA expression changes during the hibernation and reproductive periods of estrogen receptor alpha (ESR1) in Chinese alligator, Alligator sinensis.

Chinese alligator, Alligator sinensis, is a critically endangered reptile species unique to China. Little is known about the mechanism of growth- and reproduction-related hormones gene expression in Chinese alligator. Estrogens play important roles in regulating multiple reproduction- and non-reproduction-related functions by binding to their corresponding receptors. Here, the full-length cDNA of estrogen receptor alpha (ERα/ESR1) was cloned and sequenced from Chinese alligator for the first time, which comprises 1764bp nucleotides and encodes a predicted protein of 587 amino acids. Phylogenetic analysis of ESR1 showed that crocodilians and turtles were the sister-group of birds. The results of real-time quantitative PCR indicated that the ESR1 mRNA was widely expressed in the brain and peripheral tissues. In the brain and pituitary gland, ESR1 was most highly transcribed in the cerebellum. But in other peripheral tissues, ESR1 mRNA expression level was the highest in the ovary. Compared with hibernation period, ESR1 mRNA expression levels were increased significantly in the reproductive period (P<0.05) in cerebellum, pituitary gland, liver, spleen, lung, kidney and ovary, while no significant change in other examined tissues (P>0.05). The ESR1 mRNA expression levels changes during the two periods of different tissues suggested that ESR1 might play an important role in mediation of estrogenic multiple reproductive effects in Chinese alligator. Furthermore, it was the first time to quantify ESR1 mRNA level in the brain of crocodilians, and the distribution and expression of ESR1 mRNA in the midbrain, cerebellum and medulla oblongata was also reported for the first time in reptiles.

In ovo treatment with an estrogen receptor alpha selective agonist causes precocious development of the female reproductive tract of the American alligator (Alligator mississippiensis).

The molecular signaling processes involved the differentiation of the Müllerian duct (MD) into the female reproductive tract, or oviduct, in non-mammalian vertebrates are not well understood. Studies in mammals and birds indicate that steroid hormones play a role in this process, as the embryonic MD has been shown to be vulnerable to exogenous estrogens and progestins and environmental endocrine disrupting contaminants. In a previous study, developmental treatment with an estrogen receptor α (ERα) agonist, 4,4',4″-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT), induced significant enlargement of the MD in alligator embryos incubated at a male-producing temperature, which was not observed in embryos treated with an estrogen receptor ß (ERß) agonist, 7-bromo-2-(4-hydroxyphenyl)-1,3-benzoxazol-5-ol (WAY 200070), or with 17ß-estradiol (E2). In order to understand the role of estrogen signaling in female alligator oviduct development, we incubated eggs at a female-producing temperature and treated them with E2 and these ER selective agonists, PPT and WAY 200070, just prior to the thermosensitive window of sex determination. At stage 27, one stage prior to hatching, PPT induced significant enlargement of the MD with precocious development of secretory glands and connective tissue differentiation similar to characteristics of mature adult oviduct. PPT treatment in ovo increased mRNA expression of ERß, progesterone receptor, androgen receptor and insulin-like growth factor 1 in MD at stage 27, while expression of ERα was decreased. Neither WAY 200070 nor E2 treatment induced these effects seen in PPT-treated MD. The results of this study provide insight into the critical factors for healthy reproductive system formation in this sentinel species, although further investigation is needed to determine whether the observed phenomena are directly due to selective stimulation of ERα or related to some other aspect of PPT treatment.