Morphology and cytochemical patterns of peripheral blood cells of tiger frog (Rana rugulosa).

Background: Tiger frog (Rana rugulosa) is a national second-class protected amphibian species in China with an important ecological and economic value. In recent years, due to excessive human hunting, pollution and habitat loss, the wild population of tiger frog has declined sharply. To protect wildlife resources, the artificial breeding of tiger frogs has rapidly developed in China. Diseases are increasing and spreading among tiger frogs due to the increasing scale of artificial farming. The blood examination is the most straightforward and less invasive technique to evaluate the animal health condition. Thus, it is essential to obtain the normal hematological indicators of tiger frogs. The objective of this study was to investigate the morphometry, microstructure and cytochemical patterns of peripheral blood cells in tiger frogs. Methods: The number of blood cells in tiger frogs was counted on a blood count board, and the cell sizes were measured by a micrometer under light microscope. The morphology and classification of blood cells were studied by Wright-Giemsa staining, and the cytochemical pateerns was investigated by various cytochemical staining including periodic acid-Schiff (PAS), Sudan black B (SBB), peroxidase (POX), alkaline phosphatase (AKP), acid phosphatase (ACP), chloroacetic acid AS-D naphthol esterase (CAE) and α-naphthol acetate esterase (ANAE) staining. Results: Besides erythrocytes and thrombocytes, five types of leukocytes were identified in tiger frogs: neutrophils, eosinophils, basophils, lymphocytes and monocytes. The mean erythrocyte, leukocyte and thrombocyte counts were 1.33 ± 0.15 million/mm3, 3.73 ± 0.04 × 104/mm3 and 1.7 ± 0.01 × 104/mm3, respectively. Small lymphocytes were the most abundant leukocytes, followed by large lymphocytes, Neutrophils, eosinophils and monocytes, basophils were the fewest. Eosinophils were strongly positive for PAS, positive for SBB, POX, ACP, CAE, ANAE, while weakly positive for AKP staining; basophils were strongly positive for PAS, ACP, positive for SBB, CAE, weakly positive for ANAE, negative for AKP, POX staining; neutrophils were strongly positive for ACP, SBB, positive for PAS, POX, weakly positive for AKP, CAE and ANAE staining; monocytes were positive for PAS, SBB, ANAE, weakly positive for ACP, AKP, POX, CAE staining; large lymphocytes and thrombocytes were positive for PAS, ACP, weakly positive for ANAE, while negative for SBB, POX, AKP, CAE; small lymphocytes were similar to large lymphocytes, except for strongly positive for PAS and ACP staining. Conclusions: The blood cell types and morphology of tiger frogs were generally similar to those of other amphibians, while their cytochemical patterns had some notable species specificity.Our study could enrich the knowledge of peripheral blood cell morphology and cytochemistry in amphibians, and provide baseline data for health condition evaluation and disease diagnosis of tiger frogs.

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.

Hematological and cytochemical characteristics of peripheral blood cells in the argus snakehead (Ophiocephalus argus Cantor).

BACKGROUND: The argus snakehead (Ophiocephalus argus Cantor) is a highly nutritious, freshwater, cultured bony fish with a high economic value. The health of the fish is closely related to its blood cells, which are critical for oxygen transport, natural defense, and immunity. We investigated the morphometry, microstructure, and cytochemical characteristics of the peripheral blood cells of O. argus. Our results may provide the basic reference values needed to monitor the health of this fish for large-scale cultivation. METHODS: The number of blood cells in O. argus were counted on a hemocytometer and their size was measured using a micrometer under light microscope. The morphology and classification of the blood cells were studied using Wright's staining and the cytochemical characteristics were studied using seven chemical stains including peroxidase (POX), Sudan black B (SBB), periodic acid-Schiff (PAS), acid phosphatase (ACP), alkaline phosphatase (ALP), chloroacetic acid AS-D naphthol esterase (AS-D), and α-naphthol acetate esterase (α-NAE). RESULTS: The peripheral blood cells in O. argus can be classified as erythrocytes, leukocytes, and thrombocytes; of which, females had 2.9597 million/mm3, 88,400/mm3, and 43,600/mm3, respectively, and males had 3.0105 million/mm3, 105,500/mm3, and 34,000/mm3, respectively. Leukocytes consisted of neutrophils, monocytes, large lymphocytes, and small lymphocytes. Eosinophils and basophils were not found. Monocytes were the most numerous leukocytes identified, followed by neutrophils and small lymphocytes, while large lymphocytes were the least frequently identified. Cytochemical staining showed that erythrocytes were only positive for PAS staining. Neutrophils were strongly positive for POX, SBB, and ACP, and positive for all the other cytochemical stains. Monocytes were positive for PAS and α-NAE and were weakly positive for ACP and AS-D staining. Large lymphocytes were positive for PAS and were weakly positive for ALP, AS-D, and α-NAE staining. Small lymphocytes were positive for PAS and weakly positive for AS-D and α-NAE staining. Thrombocytes were positive for PAS and were weakly positive for ACP and AS-D, but negative for the remaining cytochemical stains. The morphology of peripheral blood cells in O. argus was generally similar to that of other fish species, while the cytochemical staining patterns showed clear species specificity.

Morphology and cytochemical patterns of peripheral blood cells in domestic pigeon (Columba livia).

Domestic Pigeon (Columba livia) is one of few domesticated birds with an important economic value. In this study, a comprehensive investigation on the morphology and cytochemical patterns of peripheral blood cells in domestic pigeons were conducted by using wright's and various cytochemical staining techniques including periodic acid-Schiff(PAS), sudan black B(SBB), peroxidase(POX), alkaline phosphatase(ALP), acid phosphatase(ACP), chloroacetic acid AS-D naphthol esterase(AS-D) and α-naphthol acetate esterase(α-NAE) staining. Besides erythrocytes and thrombocytes, five types of leukocytes were identified: heterophils, eosinophils, basophils, lymphocytes and monocytes. Lymphocytes were the most abundant leukocytes, followed by heterophils, eosinophils, monocytes; basophils were the fewest. Erythrocytes and thrombocytes were positive for PAS, and negative for all the other cytochemical staining. Heterophils and eosinophils exhibited positive to all cytochemical staining except for α-NAE. Basophils exhibited strongly positive for POX and AS-D, positive for PAS and ALP, while negative for SBB, ACP and α-NAE staining. Monocytes exhibited positive for PAS and α-NAE, and weakly positive for ACP, while negative for SBB, POX, ALP and AS-D staining. Lymphocytes showed positive for PAS and ACP, weakly positive for AS-D, while negative for SBB, POX, ALP and α-NAE staining. Our results add up knowledge about the domestic pigeon blood cells.

Cytochemical patterns of the peripheral blood cells in Chinese alligator (Alligator sinensis).

The Chinese alligator (Alligator sinensis) is an endemic rare crocodilian species in China. In this study, we investigated the cytochemical patterns of peripheral blood cells in Chinese alligators for the first time by a range of cytochemical staining techniques including periodic acid-Schiff(PAS), sudan black B(SBB), peroxidase(POX), alkaline phosphatase(AKP), acid phosphatase(ACP), chloroacetic acid AS-D naphthol esterase(AS-D) and α-naphthol acetate esterase(α-NAE) staining. Erythrocytes were positive for PAS, and negative for all the other staining; heterophils were strongly positive for SBB, POX, ACP, AKP, AS-D and α-NAE, while weakly positive for PAS staining; eosinophils were strongly positive for PAS, POX, AKP, ACP and AS-D, and weakly positive for SBB and α-NAE staining; basophils were strongly positive for PAS, positive for POX, ACP, AKP and α-NAE, and weakly positive for AS-D, while negative for SBB staining; monocytes were weakly positive for PAS, ACP, AKP and α-NAE, while negative for SBB, POX and AS-D staining; lymphocytes were weakly positive for PAS and α-NAE, negative for all the other staining; thrombocytes were weakly positive for PAS, and negative for all the other staining. Our results add up knowledge about Chinese alligator blood cells.

Hematology and serum biochemistry parameters of captive Chinese alligators (Alligator sinensis) during the active and hibernating periods.

The Chinese alligator Alligator sinensis is an endangered freshwater crocodilian species endemic to China. Hematology and serum biochemistry reference range are useful in the assessment and management of animal health condition. In this study, a total of 74 Chinese Alligators (30 males and 44 females) were examined to establish reference range values of hematology and serum biochemistry parameters during the active and hibernating periods. We measured and analyzed 9 hematology and 21 serum biochemistry parameters including 4 serum electrolyte parameters, and described the morphology of different types of blood cells. No statistical differences between the sexes were found for hematology parameter, while significant differences were noted for some serum biochemistry parameters, with males having greater alkaline phosphatase activity level and lower globulin concentration value than females. There were some significant differences between the two different periods with alligators during the active period possessing lower values for mean corpuscular volume, mean corpuscular hemoglobin, total bilirubin and creatine kinase, but higher values for red blood cell and white blood cell counts, monocyte percentage, aspartate aminotransferase, a-amylase, blood urea nitrogen, creatinine, triglycerides, and cholesterol. These baseline data were essential for health condition evaluation and disease diagnosis of this endangered species.

Multiple paternity: A compensation mechanism of the Chinese alligator for inbreeding.

The Chinese alligator Alligator sinensis is a critically endangered species endemic to China. Knowledge about reproductive strategies of a species contributes to their conservation. Little is, however, known about the reproductive strategies and its impact on the population. In the present study, an easy and non-invasive genetic method was used to improve the understanding of mating system of Chinese alligators and its effect on the population genetic diversity by nine polymorphic microsatellite loci. There was a high incidence of multiple paternity among 50 clutches, with a total 60% of the clutches having multiple paternity and up to three males contributing to single clutches. In addition, polyandry females choose to mate with males that are more distant in relatedness compared with monogamy females. Multiple paternity can decrease the inbreeding coefficient, while there is no significant difference between single and multiple paternity (P>0.05). Furthermore, there was an increased allelic diversity (though not heterozygosity) in multiple paternity sired offspring compared with the single paternity sired offspring in F2 generations (P<0.05), as predicted by the genetic diversity hypothesis. Multiple paternity may function as an important inbreeding avoidance compensation mechanism leading to the potential of the species to avoid extinction. These findings will not only enhance the understanding of the mating system and the biological traits of the Chinese alligator, but also improve the captive breeding program management and conservation strategies of the endangered species.

Molecular characterization and tissue expression profiles of prepro-vasoactive intestinal peptide in the Chinese alligator (Alligator sinensis) during the active and hibernating periods.

The Chinese alligator (Alligator sinensis), a freshwater crocodilian endemic to China, is one of the most endangered crocodilian species; up to this date, very little is known about the endocrine regulation of its metabolic activities during different physiological states. In this study, we characterized the structure of the prepro-vasoactive intestinal peptide in Chinese alligator (prepro-caVIP) for the first time and examined its expression profiles in various tissues during the active and hibernating periods. The prepro-caVIP cDNA consists of a 221-bp 5'-untranslated region (UTR), a 606-bp complete coding region (CDS), and a 312-bp 3'-UTR, which encodes the 201-amino acid prepro-caVIP containing a 28-amino acid vasoactive intestinal peptide (VIP) and a 27-amino acid PHI (peptide histidine isoleucine). Multiple alignment analysis showed that VIP shares 100% identity with the given birds, reptiles, and African clawed frog, and 89% identity with mammals, 96% with fishes. Real-time quantitative PCR showed that the prepro-caVIP is widely expressed in all the examined tissues, and the expression level is significantly higher in small intestine, stomach, pancreas, lung, and skeletal muscle, whereas lower in heart, liver, spleen, kidney, ovary, and oviduct. During hibernation, the expression level of caVIP was significantly decreased in small intestine (P < 0.01), pancreas, and skeletal muscle (P < 0.05), whereas significantly increased in liver, spleen, and lung (P < 0.01). The wide distribution of caVIP and its differential expression changes in various tissues during hibernation implicated that it might play multiple effects in Chinese alligator and participate in the physiological adaptation of various organs in a paracrine and/or neurocrine manner.

Immunohistochemical Localization of Somatostatin in the Brain of Chinese Alligator Alligator sinensis.

In this study, the regional distribution and histological localization of somatostatin (SS) immunoreactive (IR) perikarya and fibers was investigated for the first time in the brain of adult Chinese alligator by immunohistochemical method. The results showed SS-IR perikarya and fibers were widely distributed in various parts of the brain except for olfactory bulbs. In the telencephalon, SS-IR perikarya were predominantly located in the cellular layer and deep plexiform layer of dorsomedial and medial cortex, less in the dorsal and lateral cortex, while SS-IR fibers were found in all layers of the cerebral cortex. SS-IR perikarya and fibers were also detected in the dorsal ventricular ridge, hippocampus cortex, accessory olfactory bulb nuclearus, lenticular nucleus, and caudate nucleus. In the diencephalon, SS-IR perikarya and fibers were mainly present in supraoptic nucleus, paraventricular nucleus of hypothalamus, recessus infundibular nucleus, median eminence, the pineal gland and pituitary gland, in which the IR-fibers were abundant, appearing dot-shaped and varicosity-like. In the mesencephalon, they were present in tectum cortex, ependyma of cerebral aqueduct and the periaqueductal grey matter. Additionally, they were also detected in Purkinje's cellular layer of cerebellum, in the reticularis nucleus and raphe nucleus of medulla oblongata. The distribution pattern of SS-IR perikarya and fibers in the brain of Chinese alligator is generally similar to that reported in other reptiles, but also has some specific features. The wide distribution indicated that SS might be a neurotransmitter or neuromodulator which acts on many kinds of target cells with a wide range of physiological functions. Anat Rec, 300:507-519, 2017. © 2016 Wiley Periodicals, Inc.

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.

Mean deviation coupling synchronous control for multiple motors via second-order adaptive sliding mode control.

A new mean deviation coupling synchronization control strategy is developed for multiple motor control systems, which can guarantee the synchronization performance of multiple motor control systems and reduce complexity of the control structure with the increasing number of motors. The mean deviation coupling synchronization control architecture combining second-order adaptive sliding mode control (SOASMC) approach is proposed, which can improve synchronization control precision of multiple motor control systems and make speed tracking errors, mean speed errors of each motor and speed synchronization errors converge to zero rapidly. The proposed control scheme is robustness to parameter variations and random external disturbances and can alleviate the chattering phenomena. Moreover, an adaptive law is employed to estimate the unknown bound of uncertainty, which is obtained in the sense of Lyapunov stability theorem to minimize the control effort. Performance comparisons with master-slave control, relative coupling control, ring coupling control, conventional PI control and SMC are investigated on a four-motor synchronization control system. Extensive comparative results are given to shown the good performance of the proposed control scheme.

Speed tracking and synchronization of multiple motors using ring coupling control and adaptive sliding mode control.

A new control approach for speed tracking and synchronization of multiple motors is developed, by incorporating an adaptive sliding mode control (ASMC) technique into a ring coupling synchronization control structure. This control approach can stabilize speed tracking of each motor and synchronize its motion with other motors' motion so that speed tracking errors and synchronization errors converge to zero. Moreover, an adaptive law is exploited to estimate the unknown bound of uncertainty, which is obtained in the sense of Lyapunov stability theorem to minimize the control effort and attenuate chattering. Performance comparisons with parallel control, relative coupling control and conventional PI control are investigated on a four-motor synchronization control system. Extensive simulation results show the effectiveness of the proposed control scheme.

Follicle-stimulating hormone receptor (FSHR) in Chinese alligator, Alligator sinensis: molecular characterization, tissue distribution and mRNA expression changes during the female reproductive cycle.

The follicle-stimulating hormone (FSH) plays a central role in vertebrate reproduction, with the actions of FSH mediated by FSH receptors (FSHRs) on the granulosa cells of the ovary. The present study reports the cloning and characterization of FSHR in Chinese alligator, Alligator sinensis (caFSHR), and its tissue distribution and mRNA expression changes during the reproductive cycle. The mature protein of caFSHR displays typical features of the glycoprotein hormone receptor family, but also contains some remarkable differences when compared with other vertebrate FSHRs. The deduced amino acid sequence of the caFSHR shares identity of 85% with Chinese softshell turtle, 84-87% with birds, 77-78% with mammals, 67-73% with amphibians and 51-58% with fishes. Phylogenetic tree analysis of the FSHR amino acid sequence indicated that alligators cluster into the bird branch. Tissue expression analysis showed that caFSHR was not only expressed in the ovary, but also in the stomach, intestine, pancreas liver and oviduct at similar levels, while it was not detectable in heart, thymus or thyroid. Expression of caFSHR in the ovary is high in May (breeding prophase) and peaks in July during the breeding period, where it is maintained at high levels through September (breeding anaphase). Expression decreases significantly in November (hibernating period) and then remains relatively low from January to March (hibernating period). These temporal changes in FSHR expression suggest that it plays an important role in promoting ovarian development during the female reproductive cycle of Chinese alligator.

Molecular characterization of the Chinese alligator follicle-stimulating hormone ß subunit (FSHß) and its expression during the female reproductive cycle.

The Chinese alligator Alligator sinensis is an endangered species endemic to China, it has a highly specialized reproductive pattern with low fecundity. Up to date, little is known about the regulation of its female reproductive cycle. Follicle-stimulating hormone (FSH), a glycoprotein hormone, plays a key role in stimulating and regulating ovarian follicular development and egg production. In this study, the complete FSHß cDNA from the ovary of the Chinese alligator was obtained for the first time, it consists of 843-bp nucleotides, including 120-bp nucleotides of the 5'-untranslated region (UTR), 396-bp of the open reading frame, and 3'-UTR of 327-bp nucleotides. It encodes a 131-amino acid precursor molecule of FSHß with a signal peptide of 18 amino acids followed by a mature protein of 113 amino acids. Its deduced amino acid sequence shares high identities with the American alligator (100%) and birds (89-92%). Phylogenetic tree analysis of the FSHß amino acid sequence indicated that alligators cluster into the bird branch. Tissue distribution analyses indicated that FSHß mRNA is expressed in ovary, intestine and liver with the highest level in the ovary, while not in stomach, pancreas, heart, thymus and thyroid. Expression of FSHß in ovary increases in May (breeding prophase) and peaks in July (breeding period), it is maintained at high levels through September, then decreases significantly in November (post-reproductive period) and remains relatively low from January to March (hibernating period). These temporal changes of FSHß expression implicated that it might play an important role in promoting ovarian development during the female reproductive cycle.

Identification and localization of gastrointestinal hormones in the skin of the bullfrog Rana catesbeiana during periods of activity and hibernation.

Amphibian skin and its secretions contain a wide variety of biogenic amines and biologically active peptides, some of which are either identical or highly homologous to gastrointestinal hormones (GHs) of higher vertebrates. This study investigated the distribution density and immunoreactive (IR) intensity of 5-hydroxytryptamine (5-HT), gastrin (GAS), somatostatin (SS), pancreatic polypeptide (PP), neuropeptide Y (NPY) and glucagon (GLU) IR cells in the skin of the bullfrog Rana catesbeiana during periods of activity and hibernation. The results indicated that the six types of GHs were all present in the bullfrog skin and were most predominant in the epidermis and mucous glands. In dorsal skin, the density of the GHs-IR cells in mucous glands was higher than that in epidermis except for GAS-IR cells. In ventral skin, the density of 5-HT, PP and NPY-IR cells in mucous glands was also higher than that in the epidermis. During hibernation, the density of the six types of GHs-IR cells and the IR intensity of GAS, SS, NPY and GLU-IR cells in the epidermis of dorsal skin increased significantly. The IR intensity of SS, PP and NPY-IR cells in granular glands of ventral skin also increased significantly during hibernation. These results suggested that multiple types of GHs-IR cells present in the skin of R. catesbeiana, may play important roles in the regulation of the physiological functions of skin. Also, adaptive changes in the density and IR intensity of GHs-IR cells occurred during hibernation.

Distribution of endocrine cells in the digestive tract of Alligator sinensis during the active and hibernating period.

The digestive tract is the largest endocrine organ in the body; the distribution pattern of endocrine cells varies with different pathological and physiological states. The aim of the present study was to investigate the distributed density of 5-hydroxytryptamine (5-HT), gastrin (GAS), somatostatin (SS) and vasoactive intestinal peptide (VIP) immunoreactive (IR) cells in the digestive tract of Alligator sinensis during the active and hibernating period by immunohistochemical (IHC) method. The results indicated that 5-HT-IR cells were distributed throughout the entire digestive tract, which were most predominant in duodenum and jejunum. The density increased significantly in stomach and duodenum during hibernation. GAS-IR cells were limited in small stomach and small intestine. The density decreased significantly in small stomach during hibernation, while increased in duodenum. What's more, most of the endocrine cells in duodenum were generally spindle shaped with long cytoplasmic processes ending in the lumen during hibernation. SS-IR cells were limited in stomach and small stomach. The density increased in stomach while decreased in small stomach during hibernation, meanwhile, fewer IR cells occurred in small intestine. VIP-IR cells occurred in stomach and small stomach. The density decreased in small stomach, while increased in stomach during hibernation. These results indicated that the endocrine cells in different parts of digestive tract varied differently during hibernation, their changes were adaptive response to the hibernation.

Identification and characterization of gastrointestinal hormone immunoreactive cells in the skin and parotoids of Chinese toad Bufo gargarizans.

The skin and skin secretion of Chinese toad Bufo gargarizans have long been used in traditional Chinese medicine. However, the exact types and location of bioactive substances in Bufo gargarizans skin still have not been fully elucidated. The aim of the study was to investigate the distribution and density of six types of gastrointestinal (GI) hormone immunoreactive (IR) cells in the skin and parotoids of Bufo gargarizans. Immunohistochemistry was used for qualitative and semiquantitative analysis of GI hormone presence in the dorsal and ventral skin, and parotoids of eight adult Chinese toads. Six types of IR cells were found: serotonin (5-HT), glucagon (GLU), gastrin (GAS), somatostatin (SS), pancreatic polypeptide (PP) and neuropeptide Y(NPY) IR cells. They were mainly present in the epidermis and skin glands. 5-HT-IR cells were distributed in all layers of epidermis and glands, with higher density in the glands. Glucagon was prominently expressed in the epidermis and the bottle-shaped glands of parotoids; however, it was not present in the granular glands of skin and parotoids. The distributions of GAS and SS-IR cells were similar since they were present mainly in mucous, granular and bottle-shaped glands, while these cell types were absent in the differentiated glands of parotoids. PP-IR cells were predominant in the granular glands and the bottle-shaped glands. The expression of NPY was high in epidermal stratum granulosum and mucous glands of the dorsal skin, the bottle-shaped glands and differentiated glands of parotoids, while NPY-IR was rarely seen in the granular glands of ventral skin, and not present in the granular glands of dorsal skin and parotoids. The expression of several types of GI hormones in the skin and parotoids of Bufo gargarizans varies depending on tissue and type of glands.

Calmodulin activation of polo-like kinase 1 is required during mitotic entry.

Polo-like kinase 1 (Plk1) is a conserved key regulator of the G2/M transition, but its upstream spatiotemporal regulators remain unknown. With the help of immunofluorescence, co-immunoprecipitation, and glutathione S-transferase (GST) pull-down assay, we found that calmodulin (CaM) is one such regulatory molecule that associates with Plk1 from G2 to metaphase. More importantly, this interaction results in considerable stimulation of Plk1 kinase activity leading to hyperphosphorylation of Cdc25C. Our results provide new insight into the role of CaM as an upstream regulator of Plk1 activation during mitotic entry.

A nitroolefin functionalized BODIPY chemodosimeter for biothiols driven by an unexpected conjugated addition mechanism.

A weakly fluorescent nitroolefin functionalized BODIPY 1 was prepared and rapidly reacted with thiols through an unexpected conjugated addition to the azafulvene ring of BODIPY to generate highly fluorescent BODIPYs 4 and 5. This reaction was applied for the highly selective and sensitive detection of Cys in living cells.

Indication of intracellular physiological pH changes by L-cysteine-coated CdTe quantum dots with an acute alteration in emission color.

A novel quantum dots (QDs) based biosensor was developed to monitor physiological pH changes in both fixed and living cells by means of pH-dependent emission color of the QDs. In our system, the nominally single-sized colloidal solution samples of the L-cysteine-capped CdTe QDs with intrinsically broadened size distributions were prepared by employing aqueous synthesis technique. The quench of fluorescence intensities of the QDs with a 16 nm red shift of the emission maximum and a color change from green to yellow was observed with a slight pH decrease (from 7.0 to 6.8) in the system. This pH-dependent emission could be attributed to the efficient exciton energy transfer from smaller QDs to larger ones, which was controlled by electrostatic-tuned aggregation/disaggregation (low/high pH values) processes of the QDs. In addition to high stability, the emission shift of the QDs was reversible for at least one cycle under optimal conditions. Our pH biosensor may find potential application for monitoring the intracellular pH changes in both physiological and pathological conditions.