Identification of genes expressed in human CD34+ hematopoietic stem/progenitor cells by expressed sequence tags and efficient full-length cDNA cloning

Hematopoietic stem/progenitor cells (HSPCs) possess the potentials of self-renewal, proliferation, and differentiation toward different lineages of blood cells. These cells not only play a primordial role in hematopoietic development but also have important clinical application. Characterization of the gene expression profile in CD34⁺ HSPCs may lead to a better understanding of the regulation of normal and pathological hematopoiesis. In the present work, genes expressed in human umbilical cord blood CD34⁺ cells were catalogued by partially sequencing a large amount of cDNA clones [or expressed sequence tags (ESTs)] and analyzing these sequences with the tools of bioinformatics. Among 9,866 ESTs thus obtained, 4,697 (47.6%) showed identity to known genes in the GenBank database, 2,603 (26.4%) matched to the ESTs previously deposited in a public domain database, 1,415 (14.3%) were previously undescribed ESTs, and the remaining 1,151 (11.7%) were mitochondrial DNA, ribosomal RNA, or repetitive (Alu or L1) sequences. Integration of ESTs of known genes generated a profile including 855 genes that could be divided into different categories according to their functions. Some (8.2%) of the genes in this profile were considered related to early hematopoiesis. The possible function of ESTs corresponding to so far unknown genes were approached by means of homology and functional motif searches. Moreover, attempts were made to generate libraries enriched for full-length cDNAs, to better explore the genes in HSPCs. Nearly 60% of the cDNA clones of mRNA under 2 kb in our libraries had 5′ ends upstream of the first ATG codon of the ORF. With this satisfactory result, we have developed an efficient working system that allowed fast sequencing of 32 full-length cDNAs, 16 of them being mapped to the chromosomes with radiation hybrid panels. This work may lay a basis for the further research on the molecular network of hematopoietic regulation.     

Plasma Oxytocin and Arginine-Vasopressin Levels in Children with Autism Spectrum Disorder in China: Associations with Symptoms

Autism spectrum disorder(ASD) is defined by impairments of social interaction and the presence of obsessive behaviors. The ‘‘twin' nonapeptides oxytocin(OXT) and arginine-vasopressin(AVP) are known to play regulatory roles in social behaviors. However, the plasma levels and behavioral relevance of OXT and AVP in children with ASD have seldom been investigated. It is also unknown whether their mothers have abnormal plasma peptide levels. Here, using well-established methods of neuropeptide measurement and a relatively large sample size, we determined the plasma levels of the two neuropeptides in 85 normal children, 84 children with ASD, and 31 mothers from each group of children.As expected, children with ASD had lower plasma OXT levels than gender-matched controls(P = 0.028). No such difference was found for plasma AVP concentrations. Correlation analysis showed that ASD children with higher plasma OXT concentrations tended to have less impairment of verbal communication(Rho =-0.22,P = 0.076), while those with higher plasma AVP levels tended to have lower levels of repetitive use of objects(Rho =-0.231, P = 0.079). Unlike the findings in children, maternal plasma OXT levels showed no group difference. However, plasma AVP levels in the mothers of ASD children tended to be lower than in the mothers of normal children(P = 0.072). In conclusion, our results suggest that the OXT system is dysregulated in children with ASD, and that OXT and AVP levels in plasma seem to be associated with specific autistic symptoms. The plasma levels of OXT or AVP in mothers and their ASD children did not seem to change in the same direction.     

Staging systems for predicting survival of patients with hepatocellular carcinoma after surgery

AIM: To compare the staging systems for stratifying and predicting the prognosis of patients with hepatocel-lular carcinoma (HCC) after partial hepatectomy (PH). METHODS: Clinical data about 438 HCC patients who underwent PH from January 1991 to December 2004 at our hospital were retrospectively analyzed. Tumor stage was evaluated following the Chinese tumor node me-tastasis (TNM) and barcelona clinic liver cancer (BCLC) staging systems, respectively. Survival curves for the HCC patients were plotted using ...     

Morphine-induced conditioned place preference in rats is inhibited by electroacupuncture at 2 Hz: Role of enkephalin in the nucleus accumbens

Our previous studies have demonstrated that morphine-induced conditioned place preference (CPP) can be inhibited by 2 Hz electroacupuncture (EA). This inhibition can be blocked by either the opioid receptor antagonist naloxone (i.p.) or lesion in the nucleus accumbens (NAc), providing evidence that endogenous opioid system in the NAc mediates the effects of EA. Here we report that 1) A single session of 2 Hz EA produced a significant increase of the content of enkephalin in the NAc of morphine-induced CPP rats, and this effect was stronger in three consecutive sessions of EA; 2) Intracerebroventricular injection of the μ-opioid receptor antagonist CTAP or δ-opioid receptor antagonist NTI, but not κ-opioid receptor antagonist nor-BNI, dose-dependently reversed the inhibitory effects of 2 Hz EA on the expression of morphine-induced CPP; 3) Three consecutive sessions of 2 Hz EA up-regulated the mRNA level of preproenkephalin in the NAc of morphine-induced CPP rats. The results suggest that the inhibitory effects of 2 Hz EA on the expression of the morphine CPP is mediated by μ- and δ-, but not κ-opioid receptor, possibly via accelerating both the release and synthesis of enkephalin in the NAc. These findings support the possibility of using 2 Hz EA for the treatment of opiate addiction.     

Dynamic neuronal responses in cortical and thalamic areas during different phases of formalin test in rats

Although formalin-induced activity in primary afferent fibers and spinal dorsal horn is well described, the forebrain neural basis underlying each phase of behavior in formalin test has not yet been clarified. The present study was designed to investigate the cortical and thalamic neuronal responses and interactions among forebrain areas during different phases after subcutaneous injection of formalin. Formalin-induced neuronal activities were simultaneously recorded from primary somatosensory cortex (SI), anterior cingulate cortex (ACC) and medial dorsal (MD) and ventral posterior (VP) thalamus during different phases (i.e., first phase, interphase, second phase and third recovery phase starting from 70 min after injection) of formalin test, using a multi-channel, single-unit recording technique. Our results showed that, (i) unlike the responses in primary afferent fibers and spinal dorsal horn, many forebrain neurons displayed monophasic excitatory responses in the first hour after formalin injection, except a small portion of neurons which exhibited biphasic responses; (ii) the response patterns of many cortical and thalamic neurons changed from excitatory to inhibitory at the end of the second phase; (iii) the direction of information flow also changed dramatically, i.e., from cortex to thalamus and from the medial to the lateral pathway in the first hour, but reversed in phase 3. These results indicate that the changes of activity pattern in forebrain networks may underlie the emerging and subsiding of central sensitization-induced pain behavior in the second phase of formalin test.     

The role of NR2B containing NMDA receptor in place preference conditioned with morphine and natural reinforcers in rats

It has been reported that N-methyl-D-aspartate (NMDA) receptor is implicated in drug addiction and antagonists of the NMDA receptor complex can inhibit the development and expression of conditioned place preference (CPP) induced by several addictive drugs, implying that this class of compounds might be considered as candidate for the treatment of substance abuse. To explore this possibility, it is important to evaluate whether the inhibitory effect of NMDA receptor antagonists would be confined to behaviors produced by drugs of abuse only, but not by natural reinforcers. According to the quantitative changes of NMDA receptor subunits, including NR1, NR2A, and NR2B, induced by diverse types of reinforcers, we chose NR2B subunit as the target of research. Experimental results showed that (1) an augmented expression of NR2B subunit was revealed by Western blotting in the nucleus accumbens (NAc) and the hippocampus in rats with CPP induced by morphine, but not by natural rewards such as food, novel environment and social interaction. (2) Ifenprodil, an antagonist highly selective for NR2B subunit of the NMDA receptor, produced a dose-dependent reduction in CPP induced by morphine and novel environment, but not that by food consumption and social interaction. Taking together, these findings suggested that NR2B containing NMDA receptor may be more involved with morphine reward rather than natural rewards, and that antagonism of NR2B may have a potential for the treatment of morphine abuse.     

Contribution of the spinal cord BDNF to the development of neuropathic pain by activation of the NR2B-containing NMDA receptors in rats with spinal nerve ligation

The NMDA receptor and the brain-derived neurotrophic factor (BDNF) are involved in central sensitization and synaptic plasticity in the spinal cord. To determine whether the spinal cord BDNF contributes to the development and maintenance of neuropathic pain by activation of the dorsal horn NR2B-containing NMDA (NMDA-2B) receptors, this study was designed to investigate if alterations in BDNF and its TrkB receptor in the spinal dorsal horn would parallel the timeline of the development of neuropathic pain in lumbar 5 (L5) spinal nerve ligated (SNL) rats. The enzyme-linked immunosorbent assay (ELISA) showed that the BDNF concentration significantly increased during 24 h post-surgery, and the maximal enhancement lasted for 48 h. It declined as time progressed and returned to the level of pre-operation at 28 days after SNL. In parallel with the alteration of BDNF concentration in the spinal dorsal horn, the 50% paw withdrawal threshold (PWT) of the ipsilateral hind paw in SNL rats also showed a significant decrease during 24–48 h after SNL as compared with those in sham-operated rats. The correlation analysis revealed that the BDNF concentration had a negative correlation with 50% PWT in early stage (0–48 h) (r = -0.974, p = 0.001), but not late stage (3–28 days) (r = 0.3395, p = 0.6605), after SNL. Similarly, the immunohistochemical staining revealed that a significant up-regulation of BDNF expression in the spinal dorsal horn appeared as early as 12 h post-operation in SNL rats, peaked at 24–48 h, declined at 3 days and disappeared at 14 days after SNL. In contrast, an increase in NMDA-2B receptors expression in the spinal dorsal horn was delayed to 48 h after SNL. The increase reached peak at 3 days, lasted for 14 days, and returned to the control level of pre-operation at 28 days after SNL. The maximal enhancement of BDNF expression occurred in early stage (24–48 h) after nerve injury, while the peak of NMDA-2B receptors expression appeared in late stage (3–14 days) post-nerve ligation. As compared with the dynamic changes of 50% PWT in the timeline after nerve injury, the maximal enhancement of BDNF expression closely paralleled the maximal decline in the slope of 50% PWT, while the peak of NMDA-2B receptors expression corresponded with the plateau of the decreased 50% PWT. Therefore, the increased BDNF in the spinal dorsal horn was likely to be associated with the initiation of neuropathic pain in early stage (0–48 h), while the activation of NMDA-2B receptors was involved in the maintenance of persistent pain states in late stage (2–14 days) after nerve injury. Moreover, the present study also demonstrated that the BDNF/TrkB-mediated signaling pathway within the spinal cord might be involved in the induction of neuropathic pain in early stage after nerve injury, and the selective NMDA-2B receptors antagonist (Ro 25-6981) almost completely blocked the BDNF-induced mechanical allodynia in all of the tested rats. These data suggested that the BDNF/TrkB-mediated signaling pathway in the spinal cord was involved in the development of nerve injury-induced neuropathic pain through the activation of dorsal horn NMDA-2B receptors.     

CCK(B) receptor antagonist L365,260 potentiates the efficacy to and reverses chronic tolerance to electroacupuncture-induced analgesia in mice

Cholecystokinin octapeptide (CCK-8) is a physiological antagonist of endogenous opioids in the central nervous system (CNS). Our previous work has shown that CCK-8 plays an important role in the development of tolerance to morphine analgesia and electroacupuncture (EA) analgesia in the rat. The present studies were designed to examine whether the CCK(B) receptor is involved in the modulation of EA analgesia and the development of EA tolerance in mice. The latency to flick the tail in the radiant heat was used as index to assess the efficacy of EA analgesia. Subcutaneous (s.c.) injection of the CCK(B) receptor antagonist L365,260 produced a dose-dependent (0.125-2.0 mg/kg) potentiation of the analgesia induced by 100 Hz EA, with a maximal effect occurred at 0.5 mg/kg. In addition, L365,260 (0.5 mg/kg) significantly reversed chronic tolerance to 100 Hz EA in mice. These results suggest that the CCK(B) receptor might play a role in the tonic inhibition of 100 Hz EA-induced analgesia and in the mediation of chronic tolerance to 100 Hz EA in mice. The results opened a way for further investigation of the function of CCK-8 in pain modulation using inbred strains of mice.     

Corticofugal influences on thalamic neurons during nociceptive transmission in awake rats

Pain is a multidimensional phenomenon and processed in a neural network. The supraspinal, brain mechanisms are increasingly recognized in playing a major role in the representation and modulation of pain. The aim of the current study is to investigate the functional interactions between cortex and thalamus during nociceptive processing, by observing the pain-related information flow and neuronal correlations within thalamo-cortical pathways. Pain-evoked, single-neuron activity was recorded in awake Sprague-Dawley rats with a Magnet system. Eight-wire microarrays were implanted into four different brain regions, i.e., the primary somatosensory (SI) and anterior cingulate cortex (ACC), as well as ventral posterior (VP) and medial dorsal thalamus (MD). Noxious radiant heat was delivered to the rat hind paws on the side contralateral to the recording regions. A large number of responsive neurons were recorded in the four brain areas. Directed coherence analysis revealed that the amount of information flow was significantly increased from SI cortex to VP thalamus following noxious stimuli, suggesting that SI cortex has descending influence on thalamic neurons during pain processing. Moreover, more correlated neuronal activities indicated by crosscorrelation histograms were found between cortical and thalamic neurons, with cortical neurons firing ahead of thalamic units. On basis of the above findings, we propose that nociceptive responses are modulated by corticothalamic feedback during nociceptive transmission, which may be tight in the lateral pathway, while loose in the medial pathway.