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Biomedical subjects

L Luo

Publications and source records attributed to L Luo.

At least 109 records · Page 6Linked to original sources
PubMed1999

Distribution of the P2X2 receptor subunit of the ATP-gated ion channels in the rat central nervous system.

The distribution of the P2X2 receptor subunit of the adenosine 5'-triphosphate (ATP)-gated ion channels was examined in the adult rat central nervous system (CNS) by using P2X2 receptor-specific antisera and riboprobe-based in situ hybridisation. P2X2 receptor mRNA expression matched the P2X2 receptor protein localisation. An extensive expression pattern was observed, including: olfactory bulb, cerebral cortex, hippocampus, habenula, thalamic and subthalamic nuclei, caudate putamen, posteromedial amygdalo-hippocampal and amygdalo-cortical nuclei, substantia nigra pars compacta, ventromedial and arcuate hypothalamic nuclei, supraoptic nucleus, tuberomammillary nucleus, mesencephalic trigeminal nucleus, dorsal raphe, locus coeruleus, medial parabrachial nucleus, tegmental areas, pontine nuclei, red nucleus, lateral superior olive, cochlear nuclei, spinal trigeminal nuclei, cranial motor nuclei, ventrolateral medulla, area postrema, nucleus of solitary tract, and cerebellar cortex. In the spinal cord, P2X2 receptor expression was highest in the dorsal horn, with significant neuronal labeling in the ventral horn and intermediolateral cell column. The identification of extensive P2X2 receptor immunoreactivity and mRNA distribution within the CNS demonstrated here provides a basis for the P2X receptor antagonist pharmacology reported in electrophysiological studies. These data support the role for extracellular ATP acting as a fast neurotransmitter at pre- and postsynaptic sites in processes such as sensory transmission, sensory-motor integration, motor and autonomic control, and in neuronal phenomena such as long-term potentiation (LTP) and depression (LTD). Additionally, labelling of neuroglia and fibre tracts supports a diverse role for extracellular ATP in CNS homeostasis.

Adenosine Triphosphate↗
PubMed1999

Modulating electron density in the bound product, 4-hydroxybenzoyl-CoA, by mutations in 4-chlorobenzoyl-CoA dehalogenase near the 4-hydroxy group.

The enzyme 4-chlorobenzoyl-CoA dehalogenase hydrolyzes 4-chlorobenzoyl-CoA (4-CBA-CoA) to 4-hydroxybenzoyl-CoA (4-HBA-CoA). Biochemical and crystallographic studies have identified a critical role for the dehalogenase residue Asp 145 in close proximity to the ligand's 4-hydroxy group in the structure of the product-enzyme complex. In the present study the effects of site selective mutations at Asp 145 on the product complex are explored by Raman spectroscopy. The spectral signatures of the WT-product complex, the large red shift in lambdamax, and the complete reorganization of the benzoyl ring modes in Raman data are absent for the D145E complex. The major spectral perturbations in the WT complex are brought about by strong electron "pull" at the benzoyl carbonyl and electron "push" by the side chain of Asp 145 near the 4-OH group. Acting in concert, these factors polarize the benzoyl's pi-electrons. Since the Raman data show that very strong electron pull occurs at the benzoyl's carbonyl in the D145E complex, it is apparent that the needed electron push near the benzoyl's 4-OH group is missing. Thus, very precise positioning of Asp 145's side chain near the benzoyl's 4-position is needed to bring about the dramatic electron reorganization seen in the WT complex, and this criterion cannot be met by the glutamate side chain with its additional CH2 group. For two other Asp145 mutants D145A and D145S that lack catalytic activity, Raman difference spectroscopic data for product complexes demonstrate the presence of a population of ionized product (i.e., 4-O-) in the active sites. The presence of the ionized phenolate form explains the observation that these complexes have highly red-shifted absorbance maxima with lambdamaxs near 400 nm. For the WT complex only the 4-OH form is seen, ionization being energetically expensive with the presence of the proximal negative charge on the Asp 145 side chain. Semiquantitative estimates of the pKa for the bound product in D145S and D145A indicate that this ionization lies in the pH 6.5-7.0 range. This is approximately 2 pH units below the pKa for the free product. The Raman spectrum of 4-dimethylaminobenzoyl-CoA undergoes major changes upon binding to dehalogenase. The bound form has two features near 1562 and 1529 cm-1 and therefore closely resembles the spectrum of product bound to wild-type enzyme, which underlines the quinonoid nature in these complexes. The use of a newly developed Raman system allowed us to obtain normal (nonresonance) Raman data for the dehalogenase complexes in the 100-300 microM range and heralds an important advance in the application of Raman spectroscopy to dilute solutions of macromolecules.

Acyl Coenzyme A↗
PubMed1999

Effects of stimulus frequency and intensity on c-fos mRNA expression in the adult rat auditory brainstem.

Induction of the cellular fos gene (c-fos) is one of the earliest transcriptional changes observed following neuronal excitation. Although not an activity marker in the strict electrophysiological sense, many neurons in the central nervous system increase their c-fos expression after periods of sustained stimulation at physiological levels of intensity. In the present study, induction of c-fos mRNA expression was examined in the auditory brainstem after 1 hour of continuous free-field acoustic stimulation. Sprague-Dawley rats were exposed to pure tones of 2, 8, 16, or 32 kHz or half-octave noise bands centered on 2, 8, or 32 kHz at 80-120 dB SPL. Stimulation-induced c-fos mRNA expression was evident at all levels of the auditory brainstem, and this expression was intensity dependent. In some brain areas, induced expression manifested a clear tonotopic organization, i.e., in dorsal, posteroventral, and anteroventral cochlear nuclei, and in the medial nucleus of the trapezoid body. The inferior colliculus exhibited multiple tonotopic representations. The dorsal nucleus of the lateral lemniscus had a crude tonotopy. Although expression was present, tonotopy was not evident in periolivary nuclei or in the ventral or intermediate nuclei of the lateral lemniscus. Free-field diotic stimulation did not induce c-fos mRNA expression in the medial or lateral superior olivary nuclei. Expression was induced in the lateral superior olive by dichotic stimulation (after a unilateral cochlear ablation), and that expression was tonotopically organized. The results suggest that stimulation-induced c-fos mRNA expression can be an effective way of mapping neuronal activity in the central auditory system under both normal and pathological conditions.

Acoustic Stimulation↗
PubMed1999

Cochlear ablation alters acoustically induced c-fos mRNA expression in the adult rat auditory brainstem.

Expression of c-fos mRNA was studied in the adult rat brain following cochlear ablations by using in situ hybridization. In normal animals, expression was produced by acoustic stimulation and was found to be tonotopically distributed in many auditory nuclei. Following unilateral cochlear ablation, acoustically driven expression was eliminated or decreased in areas normally activated by the ablated ear, e.g., the ipsilateral dorsal and ventral cochlear nuclei, dorsal periolivary nuclei, and lateral nucleus of the trapezoid body and the contralateral medial and ventral nuclei of the trapezoid body, lateral lemniscal nuclei, and inferior colliculus. These deficits did not recover, even after long survivals up to 6 months. Results also indicated that neurons in the dorsal cochlear nucleus could be activated by contralateral stimulation in the absence of ipsilateral cochlear input and that the influence of the contralateral ear was tonotopically organized. Results also indicated that c-fos expression rose rapidly and persisted for up to 6 months in neurons in the rostral part of the contralateral medial nucleus of the trapezoid body following a cochlear ablation, even in the absence of acoustic stimulation. This response may reflect a release of constitutive excitatory inputs normally suppressed by missing afferent input or changes in homeostatic gene expression related to sensory deprivation. Instances of transient, surgery-dependent increases in c-fos mRNA expression in the absence of acoustic stimulation were observed in the superficial dorsal cochlear nucleus and the cochlear nerve root on the ablated side.

Acoustic Stimulation↗
PubMed1999

Anterolateral thigh flap: A review of 168 cases.

The anterolateral thigh flap based on the descending branch of the lateral circumflex femoral vessel is one of the musculocutaneous or septocutaneous flaps in the thigh. The descending branch of the lateral circumflex femoral vessel has either perforating branches or direct cutaneous branches from the intermuscular space to the anterolateral femoral skin. Since 1983, we have transferred 168 anterolateral thigh flaps for reconstruction of old burn scars, infected wounds, carcinoma excisions, for coverage of open bone fracture of the lower leg, and for congenital diseases. One hundred fifty-two cases were free flaps. The other 16 cases were pedicled flaps. The skin branches were divided into four types in our clinical series: musculocutaneous perforators (135/168 [80.4%]); intermuscular cutaneous perforators (16/168 [9.5%]); direct cutaneous branches (14/168 [8.3%]); and tiny cutaneous perforators (3/168 [1.8%]). The results were satisfactory. Only one case resulted in a failure due to tiny cutaneous branches.

Adolescent↗
PubMed1999

Effect of IL-1 beta and TNF-alpha on the expression of monocyte chemotactic protein-1 in endometriotic cells.

To investigate the clinical significance of monocyte chemotactic protein-1 (MCP-1) produced by endometriotic tissues, the endometriotic tissues were taken from 15 patients with endometriosis. MCP-1 mRNA and MCP-1 protein were determined by dot blot analysis and enzyme linked immunosorbent assay (ELISA) in endometriotic cells cultured with or without interleukin-1 beta (IL-1 beta, 2 micrograms/L), tumor necrosis factor-alpha (TNF-alpha, 20 g/L). After exposure to IL-1 beta or TNF-alpha, the expression of MCP-1 mRNA in the endometriotic cells (8.635 +/- 0.826, 7.031 +/- 0.970, respectively) were significantly higher than that in the control group (4.482 +/- 0.435, P < 0.05); The expression of MCP-1 protein in IL-1 beta and TNF-alpha group was 4.52 +/- 0.09 micrograms/L, 2.87 +/- 0.27 micrograms/L, respectively, which were significantly higher than 1.74 +/- 0.16 micrograms/L in control (P < 0.01). The results suggested that IL-1 beta and TNF-alpha could up-regulate the expression of MCP-1 in endometriotic cells, which might be related to the development of endometriosis.

Cells, Cultured↗
PubMed1999

Spatial representation of frequency in the rat dorsal nucleus of the lateral lemniscus as revealed by acoustically induced c-fos mRNA expression.

The conventional view, based largely on studies in cats, holds that the dorsal nucleus of the lateral lemniscus (DNLL) is tonotopically organized with a dorsal (low-frequency) to ventral (high-frequency) representation. Based on the topography of projections between the DNLL and inferior colliculus, it has been proposed that the rat DNLL has a concentric, inside-to-outside, tonotopic organization with high frequencies represented along the rind and low frequencies represented in the core. We used acoustic stimulation and c-fos mRNA expression to examine this issue. Results suggest that the rat DNLL does have a crude tonotopic organization and that this tonotopy has a concentric component. Following high-frequency stimulation, labeled neurons were found most frequently along the margins of DNLL, although they also tended to be more concentrated ventrally. Many fewer neurons labeled following middle-frequency stimulation, and these tended to be more uniformly distributed throughout the nucleus. Still fewer neurons labeled after low-frequency stimulation and these tended to be scattered mostly in the dorsal half of the nucleus. We conclude that: (i) many more neurons in the rat DNLL are responsive to high-frequency than to low-frequency acoustic stimulation; and (ii) that the frequency representation of the rat DNLL has both concentric and dorsal-to-ventral components.

Acoustic Stimulation↗
PubMed1999

Mosaic analysis with a repressible cell marker for studies of gene function in neuronal morphogenesis.

We describe a genetic mosaic system in Drosophila, in which a dominant repressor of a cell marker is placed in trans to a mutant gene of interest. Mitotic recombination events between homologous chromosomes generate homozygous mutant cells, which are exclusively labeled due to loss of the repressor. Using this system, we are able to visualize axonal projections and dendritic elaboration in large neuroblast clones and single neuron clones with a membrane-targeted GFP marker. This new method allows for the study of gene functions in neuroblast proliferation, axon guidance, and dendritic elaboration in the complex central nervous system. As an example, we show that the short stop gene is required in mushroom body neurons for the extension and guidance of their axons.

Animals↗
PubMed1999

Study on genital tract Chlamydia trachomatis and gonococcal infection in Han and minority (Naxi and Dai) women in China's two provinces.

OBJECTIVES: To estimate the prevalence of lower female genital tract chlamydial (GTC) and gonococcal infection in 3 different ethnic groups in China, and to analyze the risk factors influencing these infections. METHODS: A cross-sectional survey + physical and laboratory examinations. 1,800 married women aged 18-35 Han (600, from Dujiangyan city, Sichuan), Naxi (600, from Yanyuan county, Sichuan), and Dai (600, from Jinghong Prefecture, Yunnan) were investigated between March 1996 to March 1997. Information was obtained about their personal history, socioeconomic status, medical history, STDs, sexual history, contraceptive history, results of both examinations. Tabulation, t-test, Chi-square test, multiple-logistic models were used to analyze the data. RESULTS: 3.2% of Han, 12.8% of Naxi and 4.5% of Dai women tested positive for gonococci; and 9.1% of Han, 16.7% of Naxi and 27.5% of Dai women tested positive for GTC infection. The differences of GTC and gonococcal infections between Han and Naxi, and Han and Dai women were significant. CONCLUSIONS: STDs are common in China today; both GTC and gonoccocal infections are related to non-use of condom, having sexual partners other than husband, number of abortions, and low education; and condom has a function in prevention of STDs.

Adolescent↗
PubMed1999

Anatomical distribution of prolactin-releasing peptide and its receptor suggests additional functions in the central nervous system and periphery.

A recently identified neuropeptide with PRL-releasing capabilities binds to and activates a previously known orphan G protein-coupled receptor, GPR10. We initiated a study to define the pharmacology of the peptide/receptor interaction and to identify the distribution of the peptide and its receptor in the central nervous system to elucidate sites of action of the peptide. The PRL-releasing peptide (PrRP) is a C-terminally amidated, 31-amino acid peptide derived from a 98-amino acid precursor. Radioiodinated PrRP-(1-31) binds to its receptor with high affinity (1 nM) and stimulates calcium mobilization in CHOK1 cells stably transfected with the receptor. A series of N-terminal deletions reveals that the PrRP-(12-31) amino acid is equipotent to PrRP-(1-31). Further N-terminal deletions reduce the affinity of the ligand considerably, although PrRP-(25-31) is still able to compete for binding and behaves as an agonist. The arginine residues at position 26 and 30 are critical for binding, as substitution with either lysine or citrulline reduces the affinity substantially. In situ hybridization reveals a distinct tissue distribution for both the peptide and receptor messenger RNAs. The receptor is expressed abundantly in the reticular thalamic nucleus, periventricular hypothalamus, dorsomedial hypothalamus, nucleus of the solitary tract, area postrema, anterior pituitary, and adrenal medulla. The peptide messenger RNA is expressed in the dorsomedial hypothalamus, nucleus of the solitary tract, ventrolateral reticular nucleus, and intestine. This tissue distribution suggests an alternative function of PrRP than its purported hypophysiotropic function, such as a potential role for PrRP in the central feedback control of neuroendocrine and autonomic homeostasis. Further work using selective agonists and antagonists should help define additional physiological roles of this novel mammalian neuropeptide.

Animals↗
PubMed1999

Development of the Drosophila mushroom bodies: sequential generation of three distinct types of neurons from a neuroblast.

The mushroom bodies (MBs) are prominent structures in the Drosophila brain that are essential for olfactory learning and memory. Characterization of the development and projection patterns of individual MB neurons will be important for elucidating their functions. Using mosaic analysis with a repressible cell marker (Lee, T. and Luo, L. (1999) Neuron 22, 451-461), we have positively marked the axons and dendrites of multicellular and single-cell mushroom body clones at specific developmental stages. Systematic clonal analysis demonstrates that a single mushroom body neuroblast sequentially generates at least three types of morphologically distinct neurons. Neurons projecting into the (gamma) lobe of the adult MB are born first, prior to the mid-3rd instar larval stage. Neurons projecting into the alpha' and beta' lobes are born between the mid-3rd instar larval stage and puparium formation. Finally, neurons projecting into the alpha and beta lobes are born after puparium formation. Visualization of individual MB neurons has also revealed how different neurons acquire their characteristic axon projections. During the larval stage, axons of all MB neurons bifurcate into both the dorsal and medial lobes. Shortly after puparium formation, larval MB neurons are selectively pruned according to birthdays. Degeneration of axon branches makes early-born gamma neurons retain only their main processes in the peduncle, which then project into the adult gamma lobe without bifurcation. In contrast, the basic axon projections of the later-born (alpha'/beta') larval neurons are preserved during metamorphosis. This study illustrates the cellular organization of mushroom bodies and the development of different MB neurons at the single cell level. It allows for future studies on the molecular mechanisms of mushroom body development.

Animals↗
PubMed1999

Development of HIV/AIDS vaccine using chimeric gag-env virus-like particles.

We attempted to develop a candidate HIV/AIDS vaccine, by using unprocessed HIV-2 gag pr45 precursor protein. We found that a 45 kDa unprocessed HIV-2 gag precursor protein (pr45), with a deletion of a portion of the viral protease, assembles as virus-like particles (VLP). We mapped the functional domain of HIV-2 gag VLP formation in order to find the minimum length of gag protein to form VLP. A series of deletion mutants was constructed by sequentially removing the C-terminal region of HIV-2 gag precursor protein and expressed truncated genes in Spodoptera frugiperda (SF) cells by infecting recombinant baculoviruses. We found that deletion of up to 143 amino acids at the C-terminus of HIV-2 gag, leaving 376 amino acids at the N-terminus of the protein, did not affect VLP formation. There is a proline-rich region at the amino acid positions 373 to 377 of HIV-2 gag, and replacement of these proline residues by site-directed mutagenesis completely abolished VLP assembly. Our data demonstrate that the C-terminal p12 region of HIV-2 gag precursor protein, and zinc finger domains, are dispensable for gag VLP assembly, but the presence of at least one of the three prolines at amino acid positions 373, 375 or 377 of HIV-2NIH-Z is required for VLP formation. Animals immunized with these gag particles produced high titer antibodies and Western blot analyses showed that anti-gag pr45 rabbit sera react with p17, p24 and p55 gag proteins of HIV-1. We then constructed chimeric gag genes, which carry the hypervariable V3 region of HIV-1 gp120, because the V3 loop is known to interact with chemokine receptor as a coreceptor, and known to induce the major neutralizing antibodies and stimulate the cytoxic T lymphocyte responses in humans and mice. We expressed chimeric fusion protein of HIV-2 gag with 3 tandem copies of consensus V3 domain that were derived from 245 different isolates of HIV-1. In addition, we also constructed and expressed chimeric fusion protein that contains HIV-2 gag with V3 domains of HIV-1IIIB, HIV-1MN, HIV-1SF2 and HIV-1RF. The chimeric gag-env particles had a spherical morphology, and the size was slightly larger than that of a gag particle. Immunoprecipitation and Western blot analyses show that these chimeric proteins were recognized by HIV-1 positive human sera and antisera raised against V3 peptides, as well as by rabbit anti-gp120 serum. We obtained virus neutralizing antibodies in rabbits by immunizing these gag-env VLPs. In addition, we found that gag-env chimeric VLPs induce a strong CTL activity against V3 peptide-treated target cells. Our results indicate that V3 peptides from all major clades of HIV-1 carried by HIV-2 gag can be used as a potential HIV/AIDS vaccine.

AIDS Vaccines↗
PubMed1999

Amplification of human genomic DNA sequences with polymerase chain reaction using a single oligonucleotide primer.

We present two examples of exponential nucleic acid amplification with the polymerase chain reaction (PCR) in the presence of only one amplification primer. Cloning and sequencing of the PCR products generated by amplification of human genomic DNA revealed that the amplified sequence contained only one primer and its complement, at the two ends of the PCR product. Although these experiments were performed with primers derived from the sequence of the prostate specific antigen (PSA) gene and the normal epithelial cell-specific 1 gene (NES1), the amplified sequences were novel and had no homology with either PSA or NES1 DNA. While both PSA and NES1 genes reside on chromosome 19q13.3-q13.4, the amplified sequences were found by mapping to reside on chromosome 5q12 and 5p15.1-p15.3, respectively. When we examined the mechanism of amplification by PCR using one primer in these two cases, we found that there was a high homology between the PSA primer or the NES1 primer and the two regions flanking the amplified sequence of chromosome 5q12 or 5p15. This indicated that the single PSA or NES1 primer could anneal on both strands of the DNA of that region, and mediate the exponential amplification. Since this phenomenon occurred to us twice with a limited number of different PCR reactions performed in our laboratory (< 20), we believe that it may represent a common artifact of PCR. Moreover, it appears that the palindromic primer binding sites can anneal to each other forming DNA cruciforms.

Base Sequence↗
PubMed1999

[Investigation of the apoptosis and proliferation in endometriotic cells].

OBJECTIVE: To clarify whether apoptosis is involved in endometriosis and the effects of estradiol, progesterone and mifepristone on the expression of epidermal growth factor receptor(EGFR) in cultured human endometriotic cells. METHODS: Apoptosis index and bcl-2 gene expression were examined in cultured ectopic endometrial cells from women with endometriosis (n = 15 samples) and compared with eutopic endometrial cells from non-endometriosis cases(n = 11 samples). Apoptotic cells were detected by flowcytometry; bcl-2 gene expression demonstrated by in situ hybridization technique. The cultured endometriotic cells were stimulated by estradiol(E2, 10 nmol/L), progesterone(P, 100 nmol/L), and P + mifepristone(P 100 nmol/L + mifepristone 1,000 nmol/L) respectively for 5 days, the expression of EGFR mRNA of endometriotic cells were determined by reverse transcript polymerase chain reaction (RT-PCR) method. RESULTS: The apoptosis index was significantly decreased in the ectopic endometrial cells [(2.74 +/- 1.54)%] as compared to eutopic endometrial cells [(9.97 +/- 1.26)%] (P < 0.05). bcl-2 expression in endometriotic cells was significantly increased compared with the eutopic endometrial cells (P < 0.05). The expression of EGFR mRNA in E2, P group was 1.10 +/- 0.19 and 1.28 +/- 0.24 respectively, which were significantly higher than that of control(0.90 +/- 0.10, P < 0.01). In P + mifepristone group it was 0.63 +/- 0.11, significantly lower than that of control(P < 0.001). CONCLUSION: The resistance of endometriotic cells to apoptosis may be fundamental in the aetiology and (or) pathophysiology of endometriosis. Estradiol and progesterone promote hyperplasia and differentiation of endometriotic cells while mifepristone inhibits.

Adult↗
PubMed1999

[Determination of monocyte chemotactic protein-1 in cultured endometriotic cells].

OBJECTIVE: To investigate the clinical significance of monocyte chemotactic protein-1 (MCP-1) produced by endometriotic tissues. METHODS: Expressions of MCP-1 mRNA and MCP-1 protein were determined by dot blot analysis, immunohistochemical method [streptavidin biotin-peroxidase complex (SABC)] and enzyme linked immunosorbent assay (ELISA) methods in cultured endometriotic cells with median (controls) or with interleukin-1 beta(IL-1 beta) 2 ng/ml (IL-1 beta group), and tumor necrosis factor-alpha(TNF-alpha) 20 mg/ml(TNF-alpha group) respectively. The endometriotic tissues were sourced from 15 patients with endometriosis. Meanwhile, the MCP-1 protein content of cultured supernatent was also measured. RESULTS: After exposured to IL-1 beta or TNF-alpha, the expression of MCP-1 mRNA and MEP-1 protein in the endometriotic cells were significantly higher than that in the control group (P < 0.01, P < 0.05 respectively); so was the MCP-1 protein content of supernatent. CONCLUSIONS: IL-1 beta and TNF-alpha can up-regulate the expression of MCP-1 in endometriotic cells, which may be related to the development of endometriosis.

Adult↗