Nonylphenol induced apoptosis in rat testis through the Fas/FasL pathway.
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Animals↗
Biomedical subjects
X Wang
Publications and source records attributed to X Wang.
Fabrication of nano-structured porous PLLA scaffold intended for nerve tissue engineering.
Nerve tissue engineering (NTE) is one of the most promising methods to restore central nerve systems in human health care. Three-dimensional distribution and growth of cells within the porous scaffold are of clinical significance for NTE. In this study, an attempt was made to develop porous polymeric nano-fibrous scaffold using a biodegradable poly(L-lactic acid) (PLLA) for in vitro culture of nerve stem cells (NSCs). The processing of PLLA scaffold has been carried out by liquid-liquid phase separation method. The physico-chemical properties of the scaffold were fully characterized by using differential scanning calorimetry and scanning electron microscopy. These results confirmed that the prepared scaffold is highly porous and fibrous with diameters down to nanometer scale. As our nano-structured PLLA scaffold mimics natural extracellular matrix, we have intended this biodegradable scaffold as cell carrier in NTE. The in vitro performance of NSCs seeded on nano-fibrous scaffold is addressed in this study. The cell cultural tests showed that the NSCs could differentiate on the nano-structured scaffold and the scaffold acted as a positive cue to support neurite outgrowth. These results suggested that the nano-structured porous PLLA scaffold is a potential cell carrier in NTE.
Animals↗
Apoptosis in feathers of Smyth line chickens with autoimmune vitiligo.
Vitiligo is an acquired dermatological disorder characterized by a loss of epidermal melanocytes resulting in depigmentation of the skin. Mechanisms underlying the destruction of melanocytes in vitiligo remain unclear. An animal model to study spontaneously occurring autoimmune vitiligo is the mutant Smyth line (SL) of chickens. This investigation was designed to determine whether the pathogenesis of depigmentation in Smyth line chicken vitiligo (SLV) involves an apoptotic mechanism. Terminal deoxynucleotide transferase-mediated fluorescein-dUTP nick end labeling (TUNEL) was used to detect in situ cell apoptosis in cryostat sections of 2-week-old regenerating feathers. Two-week-old regenerating feathers were obtained from SL chickens and their normally pigmented controls including the parental Brown line (BL) and Light Brown Leghorn (LBL) chickens at 6, 8, 10 and 12 weeks of age. In feathers from vitiliginous SL chickens, the number of TUNEL+ cells was significantly (P<or=0.05) higher than that in the feathers of non-vitiliginous SL, BL or LBL chickens. These TUNEL+ cells were primarily located in the epithelial barb ridge where melanocyte cell bodies are located. The extent of this apoptosis in the feathers of SLV chickens varied with the severity of depigmentation of the feathers (i.e., highest in active depigmentation), suggesting a close association between apoptosis and the disappearance of melanocytes. In addition to TUNEL staining, most sections were double-stained with monoclonal antibodies specific to either CD8 or MHC class II molecules to further explore the relationship between CD8+ feather-infiltrating lymphocytes and this increase in apoptotic cells. Compared to normally pigmented controls, the number of CD8+ and MHC class II+ cells in the feather pulp and the barb ridge increased 2-4 weeks before the visible onset of SLV, and was directly related to the changes in the number of TUNEL+ cells prior to, at onset and during depigmentation. Moreover, some of these infiltrating CD8+ cells were localized next to or near the TUNEL+ cells. These observations suggest that enhanced apoptosis in the feather of SLV chickens is a pathogenic mechanism involved in the death of melanocytes and appears to be induced by infiltrating cytotoxic T lymphocytes (CD8+).
Animals↗
The deformation of an adherent leukocyte under steady shear flow: a numerical study.
Leukocyte adhesion is a pathophysiological process in which the balance between hemodynamic and adhesion forces (molecular bonds) plays a key role. In this work, we studied the deformation of an adherent leukocyte and calculated the forces exerted on it. Three model cells were proposed, considering the leukocyte as a single drop, a compound drop, and a nucleus drop, representing a cell without nucleus, a cell with a nucleus, and a nucleus only, respectively. These model cells were supposedly adherent to a smooth substrate under steady shear flow. Our numerical results showed that all three model cells deformed in function of the initial contact angle, capillary number, and Reynolds number. The single drop was the most deformable, while the nucleus drop was the most resistant to the external flow. Each of the model cells showed maximum cell deformation at a high Reynolds number. The distribution of pressure on the cell confirmed the existence of a high-pressure region downstream of the drop, which retarded further deformation of the cell and provided a positive lift force on the drop. The consideration of a highly viscous nucleus can correct the over evaluation of the cell deformation in a flow.
Capillaries↗
DNA repair gene XPD polymorphism and lung cancer risk: a meta-analysis.
Interindividual variation in lung cancer susceptibility may be modulated in part through genetic polymorphisms in the DNA repair genes, especially the genes involved in the nucleotide excision repair (NER) pathway. The xeroderma pigmentosum complementary group D (XPD) is one of the NER genes, and two of the XPD polymorphisms 751A --> C and 312G --> A have been extensively studied in the association with lung cancer, although published studies have been inconclusive. To clarify the impact of XPD polymorphisms on lung cancer risk, we performed a meta-analysis of the published data from nine (10 comparisons) individual case-control studies of 3725 lung cancer cases and 4152 controls. The results showed that individuals with the XPD 751CC genotype had a 21% (odds ratio (OR)= 1.21, 95% confidence interval (CI) = 1.02-1.43) increased risk of lung cancer compared with individuals with the 751AA genotype without any between-study heterogeneity (P = 0.26). There was also a significant association in the recessive model of 751 C allele by comparing the CC with AC + AA genotypes (OR = 1.19, 95% CI = 1.02-1.40). The results also showed a significantly increased risk of lung cancer associated with the 312AA homozygous genotype compared with the GG genotype and the 312 A allele in the recessive model (compared with GA + AA genotypes) (OR = 1.27, 95% CI = 1.04-1.56 and OR = 1.32, 95% CI = 1.09-1.59, respectively). These results support the hypothesis that both the XPD 751 C and 312 A are risk alleles and individuals with the XPD 751 CC and 312 AA genotypes are at higher risk of developing lung cancer. Large multi-center studies with precise design, and stratified/adjusted analyses of the gene-gene (haplotypes) and gene-environment interactions are needed.
DNA Helicases↗
Cloning and characterization of alpha9 subunits of the nicotinic acetylcholine receptor expressed by saccular hair cells of the rainbow trout (Oncorhynchus mykiss).
alpha9/alpha10 Subunits are thought to constitute the nicotinic acetylcholine receptors mediating cholinergic efferent modulation of vertebrate hair cells. The present report describes the cloning and sequence analysis of subunits of the alpha9-containing receptor of a hair-cell layer from the saccule of the rainbow trout (Oncorhynchus mykiss). A major alpha9 subunit, termed alpha9-I, displayed typical features of a nicotinic alpha subunit, with total coding sequence of 572 amino acids including a 16 amino-acid signal peptide. It possessed an extended cytoplasmic loop between membrane-spanning regions M3 and M4, compared with mammalian homologs. Transcript for alpha9-I was robustly expressed in the saccular hair cell layer and less prominently in trout olfactory mucosa, spleen, pituitary gland, and liver, as determined by reverse transcription-polymerase chain reaction. alpha9-I cDNA was not detected in trout brain, skeletal muscle, retina, and kidney. The alpha9-I nicotinic receptor protein was immunolocalized, with an affinity-purified antibody directed against a trout alpha9-I epitope, to hair-cell and neural sites in the saccular hair-cell layer. Foci were found at basal and basolateral membrane sites on hair cells as well as on afferent nerve. Receptor clustering was observed in hair cells bordering non-sensory epithelium. Since in higher vertebrates the alpha9 is reported to associate with another nicotinic subunit, alpha10, we examined the possibility of expression of additional nicotinic subunits in trout saccular hair cells. Message for another nicotinic subunit, termed alpha9-II, was found to be expressed in the hair cells, although more difficult to amplify than alpha9-I. In contrast to alpha9-I, alpha9-II was expressed in brain, as well as in olfactory mucosa, less prominently in pituitary gland and liver, but not in spleen, skeletal muscle, retina, or kidney. The cloned alpha9-II had a total coding sequence of 550 amino acids, which included a 17-amino-acid signal peptide, and an extended M3-M4 loop. A third nicotinic subunit message, termed alpha9-III, was PCR-amplified from trout olfactory mucosa where it was strongly expressed. However, message for alpha9-III was not detected in hair cells. Message for alpha9-III was moderately expressed in trout brain, retina, and pituitary gland but not in trout spleen, skeletal muscle, liver, and kidney. Thus, alpha9-I and alpha9-II may together contribute to the formation of the hair-cell nicotinic receptor of teleosts, where no ortholog of alpha10 appears to exist. The current work is, to our knowledge, the first description of alpha9 coding sequences directly from a vertebrate hair cell source. Further, the generality of hair cell expression of subunits for the alpha9-containing nicotinic cholinergic receptor has been extended to fishes, suggesting a similar efferent mechanism across all vertebrate octavolateralis sensory systems.
Amino Acid Sequence↗
Cortical responses to noxious stimuli during sleep.
We used magnetoencephalography to study effects of sleep on cortical responses to noxious stimuli and to clarify the mechanisms underlying pain perception. For a noxious stimulus, painful intra-epidermal electrical stimulation, which selectively activates A-delta fibers, was applied to the dorsum of the left hand. While awake, subjects were asked to count the number of stimuli silently (Attention) or ignore the stimuli (Control). During sleep, magnetic fields recorded in stage 1 sleep and stage 2 sleep were analyzed. One main component at a latency around 140-160 ms was identified in the awake condition. Multiple source analysis indicated that this main component was generated by activities in the contralateral primary somatosensory cortex (SI), bilateral secondary somatosensory cortex (SII) and insular cortex. The medial temporal area (MT) and cingulate cortex were activated later than the main component. Cortical responses in the contralateral SI, ipsilateral SII and MT, bilateral insula and cingulate cortex were significantly enhanced in Attention as compared with Control. The main component 1 M as well as later magnetic fields were markedly attenuated during sleep, suggesting that all these cortical areas are involved in pain cognition.
Attention↗
Action of kappa and Delta opioid agonists on premotor cardiac vagal neurons in the nucleus ambiguus.
Both enkephalin and dynorphin containing fibers are in close proximity to neurons in the nucleus ambiguus, including cardiac vagal neurons. Microinjection of Delta and kappa agonists into the nucleus ambiguus have been shown to evoke decreases in heart rate. Yet little is known about the mechanisms by which Delta and kappa opioid receptors alter the activity of cardiac vagal neurons. This study tests whether kappa and Delta opioid agonists can alter the activity of cardiac vagal neurons by modulating likely opioid targets including voltage gated calcium currents, and both glycinergic and GABA) neurotransmission to cardiac vagal neurons. Cardiac vagal neurons were identified in vitro by a fluorescent tracer and studied using patch clamp techniques. Neither the kappa agonist spiradoline or the Delta agonist [D-Pen(2), D-Pen(5)]enkephalin (DPDPE) modulated the voltage gated calcium currents in cardiac vagal neurons. DPDPE also did not alter either glycinergic or GABAergic synaptic neurotransmission. Spiradoline did not change GABAergic synaptic inputs, but did significantly inhibit glycinergic synaptic inputs to cardiac vagal neurons. At a concentration of 1 microM, spiradoline inhibited the amplitude of glycinergic events, and at a concentration of 5 microM, spiradoline inhibited both glycinergic amplitude and frequency. Spiradoline also inhibited both the amplitude and frequency of glycinergic miniature inhibitory post-synaptic currents, indicating kappa agonists likely act at both presynaptic and postsynaptic sites to inhibit glycinergic neurotransmission to cardiac vagal neurons.
Analgesics, Opioid↗
Intracerebroventricular administration of anti-endothelin-1 IgG selectively upregulates endothelin-A and kappa opioid receptors.
Endothelin (ET) type A receptor antagonists enhance morphine-induced antinociception and restore morphine analgesia in morphine tolerant rats [Peptides 23 (2002) 1837; Peptides 24 (2003) 553]. These studies suggest that the central ET and opioid systems functionally interact. To explore this idea further, we determined the effect of i.c.v. administration of anti-ET-1 IgG (rabbit) on brain opioid receptor and ET receptor expression. Three days after implanting cannula into the lateral ventricle, male Sprague-Dawley rats were administered 10 microl (i.c.v.) of either control rabbit IgG (2.5 microg/microl) or anti-ET IgG (2.5 microg/microl) on day 1, day 3, and day 5. On day 6, animals were killed and the caudate and hippocampus collected. Anti-ET IgG had no significant effect on expression, measured by Western blots, of mu, delta or ET-B receptors, but increased kappa opioid (59%) and ET-A (33%) receptor protein expression in the caudate. [35S]-GTP-gamma-S binding assays demonstrated that anti-ET IgG decreased [D-Ala2-MePhe4, Gly-ol5]enkephalin efficacy, but not potency in the caudate. Control experiments showed that there was no detectable rabbit IgG in caudate and hippocampal samples. These results suggest that ET in the CSF negatively regulates kappa opioid and ET-A receptors in certain brain regions. These findings support the hypothesis that CSF neuropeptides have regulatory effects and further demonstrate a link between ET and the opioid receptor system.
Animals↗
Liquid/air interfacial structure of alcohol-octyl hydroxamic acid mixtures: a study by sum-frequency spectroscopy.
The molecular structure of the liquid/air interfaces of 1-octanol, 1-decanol, n-decane and the branched decyl alcohol EXXAL 10 has been studied by sum-frequency spectroscopy (SFS) in the C-H stretching vibrational region. The data suggest that the interfaces consist of ordered molecules with closely packed alkyl tails, in close to all-trans conformation with some gauche defects. The degree of surface ordering for the branched alcohol is much higher than for octanol and decanol. When octyl hydroxamic acid (OHA) is dissolved in 1-octanol it increases the gauche conformational defects in the interfacial chains, possibly due to mixing with the surface alcohol molecules and disrupting their ordering. In contrast, we suggest that when octyl hydroxamic acid is dissolved in EXXAL 10, the surface ordering of the alcohol chains does not change. We put forward the hypothesis that the appearance of new bands, belonging to the asymmetric methylene group vibrations and to the asymmetric methyl modes in the SF spectra of the mixture suggests that the surface OHA molecules are arranged with their hydrocarbon tails tilted very close to the interface.
1-Octanol↗
Plant cytoplasm preserved by lightning.
Usually only an organism with hard parts may be preserved in the fossil record. Cytoplasm, which is a physiologically active part of a plant, is rarely seen in the fossil record. Two Cretaceous plant fossils older than 100 million years with exceptional preservation of cytoplasm are reported here. Some cytoplasm is well preserved with subcellular details while other cytoplasm is highly hydrolyzed in the cortex of the same fossil even though both of preservations may be less than 2 microm away. The unique preservation pattern, sharp contrast of preservation in adjacent cells and the exceptional preservation of cytoplasm in the cortex suggest that lightning should play an important role in the preservation of cytoplasm and that cytoplasmic membranes may be more stable than the cell contents. Interpreting the preservation needs knowledge scattering in several formerly unrelated fields of science, including geophysics, botany, biophysics, cytology and microwave fixation technology. This new interpretation of fossilization will shed new light on preservation of cytoplasm and promote cytoplasm fossils from a position of rarity to a position of common research objects available for biological research. The importance of the identification of cytoplasm in fossil lies not in itself but in how much it influences the future research in paleobotany.
Cytoplasm↗
Pathogenesis and pathology of delayed xenograft rejection in pig-to-rhesus monkey cardiac transplantation.
UNLABELLED: It has been recognized that delayed xenograft rejection (DXR) is the major barrier to the acceptance of xenotransplantation after overcoming hyperacute rejection. OBJECTIVES: This study sought to investigate the pathogenesis and pathology of delayed xenograft rejection following pig-to-rhesus monkey heart xenotransplantation. METHODS: Heterotopic xenogeneic heart transplants in the abdominal cavity were performed using piglet donors to four monkey recipients. Complete complement depletion was achieved in the recipients with repetitive doses of high-activity cobra venom factor (Y-CVF). The recipients were immunosuppressed with a combination of cyclosporine, cyclophosphamide, and steroids. Sera were analyzed for C3 and C4 levels and complement activity and anti-pig endothelial xenoantibody. The grafts were examined histopathologically and immunohistochemically for C3, C4;C5b-9, IgM, IgG, tumor necrosis factor-alpha (TNF-alpha), intercellular adhesion molecule-1(ICAM-1), CD57(NK cells), CD68 (macrophages), CD4, and CD8. RESULTS: Xenografts survived 8, 10, 13, and 13 days respectively, all developing DXR. Venous thrombosis was the outstanding feature within DXR xenografts, complicated by interstitial edema, local hemorrhage, myocardial necrosis, and mild to moderate cellular infiltration. The serum C3 levels and complement activity decreased to almost 0 from the day of transplantation due to treatment with Y-CVF. The C4 level began to decrease 2 to 4 days before the cardiac xenografts lost their function. Anti-pig endothelial xenoantibody also decreased after transplantation, slightly increasing during DXR. All rejected xenografts showed C3, C4, C5b-9, IgG, and IgM deposits to various degrees. Large numbers of macrophages (50% of total leukocytes) infiltrated the entire xenograft with a few natural killer cells (8% to 10%), as well as some CD4+ T cells (15%) and CD8+ T cells (25%). Upregulation of ICAM-1 on graft endothelial cells and TNF-alpha in the interstitium were also demonstrated in the rejected heart. CONCLUSION: Both humoral and cell-mediated immunologic reactions may play important roles in the pathogenesis of DXR. Besides C3, C4, C5b-9, IgM, and IgG destroying the xenograft, NK cells, macrophages, and CD4+ and CD8+ T cells may further aggravate the development of DXR.
Animals↗
Possible apoptotic mechanism in epidermal cell acantholysis induced by pemphigus vulgaris autoimmunoglobulins.
Through a still unclear mechanism, pemphigus vulgaris autoantibodies (PV-IgG) induce intra-epidermal acantholytic lesions responsible for severe to fatal skin wounding. We present evidence that PV lesions contain apoptotic keratinocytes, and that cell death is induced in the lesional tissue apparently before cell separation. These data suggest that apoptosis could be the cause of the acantholytic phenomenon. We show that PV-IgG and an antibody against Fas receptor (anti-FasR) induce lesions in vitro in a similar way, causing: (1) secretion of soluble FasL; (2) elevated cellular amounts of FasR, FasL (soluble and membranal), Bax and p53 proteins; (3) reduction in levels of cellular Bcl-2; (4) enrichment in caspase 8, and activation of caspases 1 and 3; (5) co-aggregation of FasL and FasR with caspase 8 in membranal death-inducing signaling complex (DISC). Hence, the Fas-mediated death signaling pathway seems to be involved in lesion formation. Moreover, we have shown that in skin organ cultures and in keratinocyte cultures, PV-IgG can induce caspase activation and DNA fragmentation, and caspase inhibitors can prevent the formation of PV-IgG-induced epidermal lesions. Altogether, these results suggest that PV-IgG-induced acantholysis may proceed through the death-signaling pathway. They highlight new perspectives on mechanisms of tissue damage in autoimmune diseases.
Acantholysis↗
Id-1 expression and cell survival.
The Id (inhibitor of differentiation or DNA binding) helix-loop-helix (HLH) proteins are a group of dominant negative regulators of basic HLH transcriptional factors which promote cell differentiation. Recent evidence has revealed that Id proteins, especially Id-1, are also able to promote cell proliferation and cell cycle progression through inactivation of tumour suppressor and activation of growth promoting pathways in mammalian cells. In addition, upregulation of Id-1 has been found in many types of human cancer and its expression levels are also associated with advanced tumour stage. Furthermore, ectopic expression of Id-1 in human cancer cells is able to induce cell proliferation under sub-optimal conditions and protect the cells against apoptosis. These lines of evidence strongly indicate Id-1 as a positive regulator of cell growth and its expression may be a key factor required for tumour cell proliferation. This review will discuss recent evidence on the role of Id-1 in cell proliferation and survival, and its significance in malignant transformation. In addition, we will highlight the recent development in the understanding of the molecular mechanisms responsible for the action of Id-1 in promoting cell survival and tumourigenesis. Finally, the therapeutic implications through inactivation of Id-1 in the treatment of human cancer will also be addressed.
Animals↗
Galectin-1 induces nuclear translocation of endonuclease G in caspase- and cytochrome c-independent T cell death.
Galectin-1, a mammalian lectin expressed in many tissues, induces death of diverse cell types, including lymphocytes and tumor cells. The galectin-1 T cell death pathway is novel and distinct from other death pathways, including those initiated by Fas and corticosteroids. We have found that galectin-1 binding to human T cell lines triggered rapid translocation of endonuclease G from mitochondria to nuclei. However, endonuclease G nuclear translocation occurred without cytochrome c release from mitochondria, without nuclear translocation of apoptosis-inducing factor, and prior to loss of mitochondrial membrane potential. Galectin-1 treatment did not result in caspase activation, nor was death blocked by caspase inhibitors. However, galectin-1 cell death was inhibited by intracellular expression of galectin-3, and galectin-3 expression inhibited the eventual loss of mitochondrial membrane potential. Galectin-1-induced cell death proceeds via a caspase-independent pathway that involves a unique pattern of mitochondrial events, and different galectin family members can coordinately regulate susceptibility to cell death.
Active Transport, Cell Nucleus↗
Allograft tolerance induced by donor apoptotic lymphocytes requires phagocytosis in the recipient.
Cell death through apoptosis plays a critical role in regulating cellular homeostasis. Whether the disposal of apoptotic cells through phagocytosis can actively induce immune tolerance in vivo, however, remains controversial. Here, we report in a rat model that without using immunosuppressants, transfusion of apoptotic splenocytes from the donor strain prior to transplant dramatically prolonged survival of heart allografts. Histological analysis verified that rejection signs were significantly ameliorated. Splenocytes from rats transfused with donor apoptotic cells showed a dramatically decreased response to donor lymphocyte stimulation. Most importantly, blockade of phagocytosis in vivo, either with gadolinium chloride to disrupt phagocyte function or with annexin V to block binding of exposed phosphotidylserine to its receptor on phagocytes, abolished the beneficial effect of transfused apoptotic cells on heart allograft survival. Our results demonstrate that donor apoptotic cells promote specific allograft acceptance and that phagocytosis of apoptotic cells in vivo plays a crucial role in maintaining immune tolerance.
Animals↗
Poor nutritional status of younger Tibetan children living at high altitudes.
BACKGROUND: The growth, development and nutrition of children in Tibet with high-altitude and unique traditional culture have recently gained attention. However, few researches are available on the nutritional status of younger children of Tibet. OBJECTIVE: The objective of the study was to evaluate the nutritional status of children below 36 months old in Tibet by means of anthropometry. DESIGN: The cross-sectional survey was conducted between August and September in 1999 and a sample of 1655 children below 36 months old was obtained using a stratified multistage cluster random sampling method from a total of seven districts of Tibet. Height, weight and hemoglobin concentration were measured and at the same time related sociodemographic and environmental information were collected. NCHS/CDC/WHO reference data were used to evaluate the nutritional status of the entire study children population and estimate the prevalence of stunting, underweight and wasting. RESULTS: Compared with the reference of NCHS/CDC/WHO, the distributions of HAZ and WAZ shifted to the left significantly and the means of HAZ and WAZ were -1.53 and -1.05, respectively, which were significantly lower than the reference value. The distribution of WHZ, however, was close to that of the reference. The reduction of Z-scores for height and weight occurred very early in life and was greatest in the second year after birth. The prevalence of malnutrition of children was 39.0% for stunting, 23.7% for underweight and 5.6% for wasting, respectively. Rural children had prevalence of stunting of 41.4% and underweight of 24.7%, as compared with prevalence of stunting of 25.3% and underweight of 18.1% for urban children. Stunting and underweight were associated with altitudes. The mean of hemoglobin (Hb) of children was 120.4 g/l and rural children had a significantly lower Hb concentration (119.9 g/l) than urban children (123.3 g/l). The prevalence of anemia seemed higher when using different altitude corrections for Hb to estimate the prevalence, but the consequences were uneven. CONCLUSIONS: For Tibetan young children, the nutritional status of the entire population is poor and the prevalence of malnutrition is higher, especially for stunting. Malnutrition is related to high altitudes. Although the Hb concentration is higher induced by high altitude, there must be quite a lot of anemic children at high altitudes. The relationship between altitude and Hb for children on the Tibetan plateau requires further study in order to determine correctly the magnitude of anemia of children.
Altitude↗
CMV enhancer/human PDGF-beta promoter for neuron-specific transgene expression.
Using cell-type-specific promoters to restrict expression of therapeutic genes to particular cells is an attractive approach for gene therapy, but often hindered by inefficient transcriptional activities of the promoters. Knowing the enhancer for the human cytomegalovirus (CMV) immediate-early gene improves activities of several cell-type- or tissue-type-specific promoters, we set out to investigate whether it improves neuronal transgene expression driven by a neuron-specific promoter, the platelet-derived growth factor B-chain (PDGF-beta) promoter. A hybrid promoter was constructed by appending a 380-bp fragment of the CMV enhancer 5' to the PDGF-beta promoter. The plasmid containing the promoter was complexed with polyethylenimine for in vitro and in vivo gene transfer. In cultured cells, the plasmid with the hybrid promoter significantly augmented expression of a luciferase reporter gene, providing expression levels 8- to 90-fold and 7- to 178-fold higher than those from two baseline constructs containing the PDGF-beta promoter alone and the CMV enhancer alone, respectively. In particular, the activities of the hybrid promoter in two neural cell lines were close to or higher than that of the CMV immediate-early gene enhancer/promoter, a transcriptional control element that has been considered to be the most robust one identified thus far. After stereotaxic injection into the hippocampus and striatum in rats, the hybrid promoter displayed a neuronal specificity, driving gene expression almost exclusively in neurons. Transgene expression in the brain driven by the hybrid promoter was detectable 24 h after injection, being 10-fold higher than that driven by the PDGF-beta promoter alone. The expression peaked around 5 days at 1.5 x 10(5) relative light units per brain and lasted for at least 4 weeks. This differed strikingly from the expression driven by the PDGF-beta promoter, which was no longer detectable on day 3. The new gene regulatory construct reported in this study will be useful to improve neuronal transgene expression required for gene therapy of neurological disorders and functional studies of the nervous system.
Animals↗