[Effect of nitric oxide on the preterm neonatal rabbit ductus arteriosus cystathionine-γ-lyase/hydrogen sulfide pathway].

OBJECTIVE: To investigate the impact of sodium nitroprusside (a nitric oxide donor) in the ductus arteriosus in preterm rabbits on hydrogen sulfide (H(2)S)-cystathionine-γ-lyase (CSE) system. METHODS: For 16 Japanese white rabbits pregnant for 21 days were randomly divided into four groups, each of the following groups had 4 rabbits: control group, intraperitoneal injection of sodium nitroprusside 1 mg/kg, 2.5 mg/kg, and 5.0 mg/kg groups. The rabbits in control group had a peritoneal puncture with a simple hollow needle, and those in the other groups were given corresponding dose of intraperitoneal injection of sodium nitroprusside at gestational age 23 and 25 days, respectively. At gestational age 26 days the fetuses of the pregnant rabbits were removed surgically, and 28 fetal rabbits were obtained from the control group, 27 from the sodium nitroprusside small dose group, 29 from the medium dose group, and 26 from the large dose group. The fetal heart blood sample of 1 ml was taken from each fetus, and immediately after sampling the arterial ductal tissues were dissected. Fetal rabbit plasma proteins hydrogen sulfide content was determined by using de-protein method, and real time quantitative RT-PCR was used for determination of arterial tissue CSE gene and western-blotting was used for measuring protein expression of CSE. RESULTS: In control group hydrogen sulfide content of fetal rabbits plasma (55.68 ± 6.57) µmol/L and arterial tissue CSE mRNA expression was 1.07 ± 4.12; the parameters in intraperitoneal injection of sodium nitroprusside group 1 mg/kg were (60.02 ± 6.09) µmol/L and 3.46 ± 0.18; in intraperitoneal injection of sodium nitroprusside group 2.5 mg/kg, were (64.71 ± 7.12) µmol/L and 10.95 ± 0.22; and in intraperitoneal injection of sodium nitroprusside group 1 mg/kg were (70.63 ± 8.07) µmol/L and 19.56 ± 0.17. Comparison between small dose group and control group, medium dose group and small dose group, high dose group and medium dose group showed that the above data were significantly different P < 0.05, with the injection of sodium nitroprusside CSE protein expression increased gradually with increasing doses. CONCLUSION: Sodium nitroprusside showed an enhancing effect on preterm CSE-H(2)S system in rabbit ductus arteriosus in a certain range of concentration in a dose-dependent manner.

Epidermal neural crest stem cell (EPI-NCSC)--mediated recovery of sensory function in a mouse model of spinal cord injury.

Here we show that epidermal neural crest stem cell (EPI-NCSC) transplants in the contused spinal cord caused a 24% improvement in sensory connectivity and a substantial recovery of touch perception. Furthermore we present a novel method for the ex vivo expansion of EPI-NCSC into millions of stem cells that takes advantage of the migratory ability of neural crest stem cells and is based on a new culture medium and the use of microcarriers. Functional improvement was shown by two independent methods, spinal somatosensory evoked potentials (SpSEP) and the Semmes-Weinstein touch test. Subsets of transplanted cells differentiated into myelinating oligodendrocytes. Unilateral injections of EPI-NCSC into the lesion of midline contused mouse spinal cords elicited bilateral improvements. Intraspinal EPI-NCSC did not migrate laterally in the spinal cord or invade the spinal roots and dorsal root ganglia, thus implicating diffusible factors. EPI-NCSC expressed neurotrophic factors, angiogenic factors, and metalloproteases. The strength of EPI-NCSC thus is that they can exert a combination of pertinent functions in the contused spinal cord, including cell replacement, neuroprotection, angiogenesis and modulation of scar formation. EPI-NCSC are uniquely qualified for cell-based therapy in spinal cord injury, as neural crest cells and neural tube stem cells share a higher order stem cell and are thus ontologically closely related.

Norepinephrine transport-mediated gene expression in noradrenergic neurogenesis.

BACKGROUND: We have identified a differential gene expression profile in neural crest stem cells that is due to deletion of the norepinephrine transporter (NET) gene. NET is the target of psychotropic substances, such as tricyclic antidepressants and the drug of abuse, cocaine. NET mutations have been implicated in depression, anxiety, orthostatic intolerance and attention deficit hyperactivity disorder (ADHD). NET function in adult noradrenergic neurons of the peripheral and central nervous systems is to internalize norepinephrine from the synaptic cleft. By contrast, during embryogenesis norepinephrine (NE) transport promotes differentiation of neural crest stem cells and locus ceruleus progenitors into noradrenergic neurons, whereas NET inhibitors block noradrenergic differentiation. While the structure of NET und the regulation of NET function are well described, little is known about downstream target genes of norepinephrine (NE) transport. RESULTS: We have prepared gene expression profiles of in vitro differentiating wild type and norepinephrine transporter-deficient (NETKO) mouse neural crest cells using long serial analysis of gene expression (LongSAGE). Comparison analyses have identified a number of important differentially expressed genes, including genes relevant to neural crest formation, noradrenergic neuron differentiation and the phenotype of NETKO mice. Examples of differentially expressed genes that affect noradrenergic cell differentiation include genes in the bone morphogenetic protein (BMP) signaling pathway, the Phox2b binding partner Tlx2, the ubiquitin ligase Praja2, and the inhibitor of Notch signaling, Numbl. Differentially expressed genes that are likely to contribute to the NETKO phenotype include dopamine-beta-hydroxylase (Dbh), tyrosine hydroxylase (Th), the peptide transmitter 'cocaine and amphetamine regulated transcript' (Cart), and the serotonin receptor subunit Htr3a. Real-time PCR confirmed differential expression of key genes not only in neural crest cells, but also in the adult superior cervical ganglion and locus ceruleus. In addition to known genes we have identified novel differentially expressed genes and thus provide a valuable database for future studies. CONCLUSION: Loss of NET function during embryonic development in the mouse deregulates signaling pathways that are critically involved in neural crest formation and noradrenergic cell differentiation. The data further suggest deregulation of signaling pathways in the development and/or function of the NET-deficient peripheral, central and enteric nervous systems.

An epidermal neural crest stem cell (EPI-NCSC) molecular signature.

Here, we report the first transcriptome for mouse epidermal neural crest stem cells (EPI-NCSC, formerly eNCSCs). In addition, our study resolves conflicting opinions in the literature by showing that EPI-NCSC are distinct from other types of skin-resident stem cells/progenitors. Finally, with the three gene profiles, we have established a foundation and provide a valuable resource for future mouse NCSC research. EPI-NCSC represent a novel type of multipotent adult stem cell that originates from the embryonic neural crest and resides in the bulge of hair follicles. We performed gene profiling by LongSAGE (long serial analysis of gene expression) with mRNA from EPI-NCSC, embryonic NCSC, and in vitro differentiated embryonic neural crest progeny. We have identified important differentially expressed genes, including novel genes and disease genes. Furthermore, using stringent criteria, we have defined an NCSC molecular signature that consists of a panel of 19 genes and is representative of both EPI-NCSC and NCSC. EPI-NCSC have characteristics that combine advantages of embryonic and adult stem cells. Similar to embryonic stem cells, EPI-NCSC have a high degree of innate plasticity, they can be isolated at high levels of purity, and they can be expanded in vitro. Similar to other types of adult stem cell, EPI-NCSC are readily accessible by minimal invasive procedure. Multipotent adult mammalian stem cells are of great interest because of their potential value in future cell replacement therapy by autologous transplantation, which avoids graft rejection.

Characterization of epidermal neural crest stem cell (EPI-NCSC) grafts in the lesioned spinal cord.

We have characterized in the contusion-lesioned murine spinal cord the behavior of acutely implanted epidermal neural crest stem cells (EPI-NCSC, formerly eNCSC). EPI-NCSC, a novel type of multipotent adult stem cell, are remnants of the embryonic neural crest. They reside in the bulge of hair follicles and have the ability to differentiate into all major neural crest derivatives (Sieber-Blum, M., Grim, M., Hu, Y.F., Szeder, V., 2004. Pluripotent neural crest stem cells in the adult hair follicle. Dev. Dyn. 231, 258-269). Grafted EPI-NCSC survived, integrated, and intermingled with host neurites in the lesioned spinal cord. EPI-NCSC were non-migratory. They did not proliferate and did not form tumors. Significant subsets expressed neuron-specific beta-III tubulin, the GABAergic marker glutamate decarboxylase 67 (GAD67), the oligodendrocyte marker, RIP, or myelin basic protein (MBP). Close physical association of non-neuronal EPI-NCSC with host neurites was observed. Glial fibrillary acidic protein (GFAP) immunofluorescence was not detected. Collectively, our data indicate that intraspinal EPI-NCSC demonstrate several desirable characteristics that may include local neural replacement and re-myelination.

[A novel gene involved in blood glucose regulation].

OBJECTIVE: To clone a novel gene relative to blood glucose regulation. METHODS: Rat modes of autonomous regulation of blood glucose was made by intra jugular vein to right atrium injection of high concentration of glucose solution, and the control rats were injected with 0.9%NaCl both before skeletal muscles were separated for gene analysis. The differentially expressed fragments were identified by differential display technology (DDRT-PCR). After slot blot and Northern blot analysis, the artificial positive fragments were excluded and the true EST (expression sequence tag) differentially expressed was obtained. These positive EST were used as probes to screen cDNA library of rat skeletal muscle. RESULTS: A novel full-length cDNA, named as Fang-2 was obtained. GenBank Accession No. was AF399874. Fang-2 was found rat homologue of human troponin T by blast software (NCBI). It shared 78% identical nucleotides, which showed the family proteins were conservative. After high concentration of glucose stimulation of rats, the expression of Fang-2 was down-regulated. CONCLUSIONS: A novel gene relative to blood glucose regulation was cloned from rat skeletal muscle. The gene can regulate blood glucose level by effect certain mechanisms unknown yet with down-regulation expression.

[Serial analysis of gene expression in the pituitary adenomas and para-tumor normal pituitary tissues].

OBJECTIVE: To observe the characteristics and difference of gene expression in the pituitary adenomas and para-tumor normal pituitary tissues. METHODS: Using serial analysis of gene expression (SAGE), two SAGE libraries were generated. Forty clones from each SAGE library were sequenced, and the results were analyzed by SAGE2000 software and compared with the SAGE map at NCBI. RESULTS: A total of 655 gene tags, representing 43 genes, were extracted from the 40 sequence files of the para-tumor normal pituitary tissues and 737 gene tags, representing 53 genes, were extracted from the 40 sequence files of the pituitary adenomas. Of these tags, 13 were not reported before. The genes related to pituitary hormone secretion and energy metabolism were highly expressed in the two kinds of tissues. Some growth factors and cytokines were also expressed, including those involved in the immunological system. But there were also much difference of gene expression in the two tissues. Thirty-one and five tags were only detected in para-tumor normal pituitary tissues and pituitary adenomas, respectively. CONCLUSIONS: Genes involved in hormones secretion and energy metabolism were highly expressed in the pituitary adenomas and para-tumor normal pituitary tissues. Many growth factors and cytokines were also expressed in pituitary. There was also much difference of gene expression in the two kinds of tissues. SAGE can be used not only in understanding the quantity information of gene expression, but also in finding new genes.