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Publications and source records attributed to L Xiao.
Successful extraction of DNA from formalin-fixed Paraffin embedded tissues is important to DNA typing for forensic purpose. In the research, 26 samples were analyzed by method of PM and HLA-DQA1 loci typing. 24 of these samples were successful. It showed that this technique and method could be applied to individual identification and paternity test by use of these sorts of samples.
The superinduction of spermidine/spermine N1-acetyltransferase (SSAT) gene has been associated with a cytotoxic response to a new class of antineoplastic polyamine analogues. The initial mechanism of SSAT superinduction is an increase in transcription in response to analogue exposure. This increased transcription appears to be modulated through the association between a nuclear protein factor and a cis-element described here as the polyamine-responsive element (PRE). The PRE was identified as a 9-base pair sequence, 5'-TATGACTAA-3', in the context of a 31-base pair stretch from -1522 to -1492 base pairs with respect to the SSAT transcriptional start site. This element binds a nuclear factor from polyamine analogue-responsive cells, but not from polyamine analogue-insensitive cells. The labeled PRE was used to clone and identify the transcription factor, Nrf-2, that binds constitutively to the PRE sequence. Although the PRE sequence shares homology to the originally identified Nrf-2 recognition sequence, the two sequences are not identical. The Nrf-2 transcription factor appears only to be present in cell types that are capable of expressing high amounts of SSAT. The results of these studies suggest that Nrf-2, bound to the PRE, plays an important regulatory role of expression of the human SSAT gene.
OBJECTIVE: To study coreceptor usage of sequential primary HIV-1 isolates in a longitudinal follow-up cohort of HIV-1-infected men to understand its contribution to pathogenesis of HIV disease. DESIGN: Viral coreceptor usage of sequential primary isolates from HIV-1-infected individuals was examined at various timepoints and data was compared with CD4 cell counts, rates of disease progression and beta-chemokine production. METHODS: Fifty-eight sequential primary isolates were obtained from four rapid progressors, six late progressors, and three long-term nonprogressors (LTNP) and their coreceptor usage was examined by infection of peripheral blood mononuclear cells (PBMC) from donors with wild-type or non-functional CC-chemokine receptor (CCR)-5, and by infection of GHOST4 cells expressing CD4 and various chemokine receptors [CCR-1-CCR-5, CXC-chemokine receptor (CXCR)-4, BOB/GPR15, BONZO/STRL33]. Production of RANTES and macrophage inflammatory protein (MIP)-1beta was examined using unstimulated or phytohemagglutinin (PHA)-stimulated PBMC isolated from these individuals at multiple timepoints during infection. RESULTS: A switch from single CCR-5 coreceptor usage to multiple coreceptor usage occurred in all four rapid progressors and three out of six late progressors. In addition to the commonly used coreceptors CXCR-4, CCR-5, and CCR-3, some of the viruses isolated from patients in the terminal stage of infection also used CCR-1, CCR-2b, CCR-4, and BOB as coreceptors. The emergence of viral variants capable of utilizing multiple coreceptors generally preceded CD4 cell decline to < 200 x 10(6)/l and correlated with the onset of AIDS. In contrast, three LTNP maintained exclusive usage of CCR-5 over a period of 7-12 years post-infection. Endogenous production of RANTES and MIP-1beta by PBMC from LTNP was not significantly different from rapid and late progressors. However, PHA-driven production of both chemokines was significantly higher in LTNP, suggesting that in vivo activating stimuli might curtail HIV replication by inducing these chemokines. CONCLUSIONS: Viral variants capable of utilizing a broad range of coreceptors correlated with HIV-1 disease progression. In contrast, LTNP maintain exclusive usage of CCR-5 and produce higher levels of beta-chemokines. Thus, both viral and host determinants leading to the emergence of viral variants capable of using an expanded range of coreceptors may be likely determinants of disease progression.
We quantified and compared the rates at which chloroethylclonidine (CEC) inactivated cloned rat alpha1A, alpha1B-, and alpha1D-adrenoceptors. Membranes from cells transfected with one of the three cloned alpha1-adrenoceptors were incubated for various intervals with 100 microM chloroethylclonidine at 10 degrees C, 25 degrees C or 37 degrees C. The fraction of receptors alkylated by chloroethylclonidine was determined by [3H]prazosin binding. Chloroethylclonidine fully inactivated each alpha1-adrenoceptor subtype via a first order reaction. Alkylation by chloroethylclonidine was markedly slower for the alpha1A-adrenoceptor vs. the other two subtypes (rate constants in 10(-3) min(-1) at 10 degrees C: 0.99 +/- 0.01 (alpha1A), 7.26 +/- 0.15 (alpha1B), and 7.01 +/- 0.12 (alpha1D)). Despite differences in rate, activation energies for alkylation were similar among subtypes. suggesting a similar binding sites for chloroethylclonidine. Computer simulations of kinetic data in mixed receptor populations and experiments with membranes from rat brain showed that nonlinear curve fitting could distinguish relative proportions of alpha1A-adrenoceptor vs. the other two subtypes. We conclude that measurement of the rate of alkylation by chloroethylclonidine, rather than the total amount of alkylation, is most useful in distinguishing the relative proportion of alpha1A-adrenoceptor in tissues.
The relevance of a TNF-alpha promoter polymorphism, a G-to-A polymorphic sequence at position-308, was examined to test whether variant alleles of TNF-alpha affect susceptibility to infection with HIV-1 and progression to AIDS. Analysis of specimens from cohorts of HIV-1 positive homosexual men demonstrated that 3 of the 32 (9.4%) HIV-1-infected long-term nonprogressors (LTNPs) were homozygous for the uncommon TNF-2 allele compared with 3 of the 196 (1.5%) HIV-1-seronegative blood donors and uninfected homosexual men (p < 0.05). There was no difference in heterozygosity among HIV-1-seropositive or -seronegative groups, although some of the seropositive men heterozygous for the TNF2 genotype were also heterozygous for CCR5delta32. However, no significant association was found between TNF genotypes and time of survival, CD4 slopes, or viral loads when seroincident (n = 109) and seroprevalent cases (n = 442) from the Chicago MACS were analyzed. Functional analysis of lymphocytes from the seronegative group revealed no difference in endogenous or mitogen-induced TNF-alpha production, as well as susceptibility to in vitro HIV-1 infection between different TNF-genotype donors. These data suggest that TNF genotypes do not play a direct role in HIV-1 disease progression; however, they could potentially be part of a multigenic linkage that may be involved in delaying progression to AIDS.
The cellular tropism of human immunodeficiency virus type 1 (HIV-1) is dependent on utilization of specific chemokine co-receptor: macrophage-tropic/non-syncytium-inducing (NSI) viruses use CCR5, whereas T-cell tropic/syncytium-inducing (SI) viruses preferentially use CXCR4. We have analyzed co-receptor usage of 24 phylogenetically distinct primary HIV-1 isolates representing group M (clades A-F) and group O with known SI and NSI phenotype, using lymphocytes from donor with nonfunctional CCR5 (CCR5-/-; homozygous 32-bp deletion). While all SI isolates infected CCR5-/- lymphocytes (and hence do not require CCR5 for viral entry), all NSI isolates, regardless of clade, did not infect CCR5-/- lymphocytes. Thus, CCR5 expression is required for infection with NSI isolates and the CCR5 usage is independent of viral genotype. To localize the viral determinant involved in CCR5 binding, the V3 sequences across the clades were aligned based on the CCR5 usage. There were conserved uncharged residues at position 11 of V3 (mostly serine/glycine) and negatively charged residues at residue 25 (mostly glutamic/aspartic acid) among all isolates that used CCR5, whereas substitution with arginine or glutamine at these two positions led to usage of a co-receptor other than CCR5. This analysis led us to identify a consensus motif S/GXXXGPGXXXXXXXE/D within the V3 loop that predicts CCR5 co-receptor usage. Most isolates, with exception of one isolate, containing the conserved motif and predicted to utilize CCR5 indeed had an absolute requirement of CCR5 expression for infectibility. Site-directed mutagenesis in the infectious molecular clone further confirmed these results. Taken together, these data provide evidence that sequences within the V3 loop provide important residues that might be directly or indirectly involved in binding to a CCR5 co-receptor.
Based upon the observation that estrogen acts in the striatum to rapidly modulate dopamine (DA) neural transmission and DA-mediated behaviors, it has been postulated that these effects of estrogen are mediated by a specific, membrane-bound receptor mechanism. To further characterize the pharmacological specificity of the estrogen binding site, the present experiments examine effects of various estrogen agonists on amphetamine (AMPH)-induced DA release from striatal tissue of ovariectomized female rats, using a superfusion method. Catechol estrogens 4-, and 2-hydroxyestradiol, but not 2-methoxyestradiol, significantly enhance AMPH-induced striatal DA release. Estrogen metabolites, estrone and estriol, and the non-steroidal estrogen analog, diethylstilbestrol, are without effects. Estradiol conjugated to bovine serum albumin (BSA) mimics the effect of estradiol to enhance stimulated striatal DA release. These results indicate that the steroidal configuration and hydroxylation on the A-ring of estrogenic compounds may be important determinants of ligand binding to the putative estrogen binding site in the striatum. Furthermore, the effectiveness of the estradiol conjugated to BSA reinforces the idea of an external membrane-bound receptor binding site in the striatum.
1. In this study, we have cloned, expressed, and characterized an alpha1a-adrenoceptor gene from the mouse. We designed oligonucleotide PCR primers complementary to regions of the rat alpha1a-adrenoceptor sequence and amplified cDNA fragments from total RNA of mouse cerebral cortex, liver and kidney by reverse transcription-polymerase chain reaction (RT PCR). 2. Both the nucleotide and deduced peptide sequences of the cDNA showed high sequence identity with those of cloned alpha1a-adrenoceptors from other species. The cDNA clone had an open reading frame of 1398 nucleotides encoding a 466 amino acid peptide which had 97%, 92% and 90% identity with the deduced amino acid sequences of the rat, human and bovine alpha1a-adrenoceptor, respectively. 3. The amplified mouse cDNA was inserted into a mammalian expression vector pcDNA3.1(+) and expressed in COS-1 cells. The pharmacological properties of the mouse cDNA clone were examined in radioligand binding studies and functional assays. The expressed mouse protein had a high affinity for [3H]-prazosin (Kd = 0.48 nM) and pattern of affinity for antagonists in competition studies that is similar to that of the rat alpha1a-adrenoceptor. Chloroethylclonidine (CEC) could slowly alkylate the expressed protein, with a rate similar to that of the rat alpha1a-adrenoceptor. 4. The expressed receptors were able to mediate noradrenaline (NA) stimulation of the production of inositol phosphates in COS-1 cells, consistent with coupling to phospholipase C. This response to NA could be reversed by pretreatment of the transfected cells with prazosin. 5. Based on the above evidence, we concluded that the cloned cDNA is that of the mouse alpha1a-adrenoceptor.
The glucose transporter of human erythrocytes (Glut1) was reconstituted into soybean phospholipid liposomes by a method of direct incorporation using the nonionic detergent n-octyl beta-D-glucopyranoside. The reconstituted proteoliposomes were proved to be intact and low ionic permeability. Freeze-fracture electron microscopy study showed that the diameter of the proteoliposomes was about 150 +/- 50 nm and the protein was randomly distributed. The kinetic parameters of the reconstituted transporter were: Km =16.23 mmol/L, Vmax = 34.48 nmol/sec x mg protein. Furthermore, about 90% of the glucose transporter in the reconstituted proteoliposomes were orientated inside-out. Until now it is a more efficient method for uni-directional reconstitution of Glut1 with good reproducibility and higher transport activity.
Because malaria-stimulated cytokine production may have deleterious effects on human immunodeficiency virus type 1 (HIV-1) replication, the effects of Plasmodium falciparum antigens on HIV-1 replication were studied. Stimulation with malarial antigens significantly enhanced HIV-1 replication of HIV-1LAV and primary HIV-1 isolates (subtype A) in CD8-depleted peripheral blood mononuclear cells from naive donors. The malarial antigen-induced activation of HIV-1 was due to cellular activation as judged by the expression of cell activation markers and proliferative responses. While malarial antigen stimulation increased expression of tumor necrosis factor (TNF-alpha) and interleukin-6 (IL-6), only monoclonal antibodies (MAbs) to TNF-alpha inhibited malarial antigen-induced HIV-1 replication, whereas MAb to IL-6 had no effect. Malarial antigen increased HIV-1 replication by increasing viral mRNA expression and by activating long terminal repeat-directed viral transcription. These data suggest that P. falciparum infection can modulate HIV-1 pathogenesis by activating lymphocytes and stimulating viral replication through the production of cytokines.
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The chromosomal telomeric association in peripheral blood lymphocytes from 25 untreated patients with nasopharyngeal carcinoma (NPC) and in NPC cell lines (CNE) was investigated by using chromosome G banding technique. The cell telomeric association rate of patients (35.07%) was found to be higher than that of controls(20.27%) (P < 0.01), while in the two groups the distributions of chromosomes involved were approximately the same (P > 0.05). The telomeric association rates of patients were significantly higher than those of control in chromosomes 1, 2, 3, 4, 7, 11, 15, and 17(P < 0.05). In NPC lines (CNE), 94% of the cell division phases showed telomeric association. Chromosomes 2, 3, 5, 11 and 16 had higher frequencies of association than other chromosomes. The most frequent telomeric association was single chromatid association.
In order to study the mechanism of cholesterol gallstone formation through rabbit model which was induced by high cholesterol diet, we investigated the LDL receptor activity of hepatocytes binding to 125I-LDL in different phases, namely 1, 2, 3 week group and 4 week, and in a control group besides. In this animal experiment, cholesterol gallstones were induced at 2 week, 3 week and 4 week groups in 4/10, 6/10, and 7/10 cases respectively. The Bmax values of LDL receptor of hepatocytes binding to 125I-LDL decreased significantly in 3 week and 4 week groups (vs 1 week and control groups, P < 0.05). The kd values became increased in 3 week and 4 week groups (vs 1 week and control group, P < 0.05), which suggested that the activity of LDL receptor decreased gradually. In conclusions owing to the intake of high cholesterol diet with the passage of time, the decreased activity of LDL receptor of hepatocytes would reduce the synthesis of bile acid.
Using cytological examination, the cerebrospinal fluid(CSF) of 98 patients with acute viral encephalitis was investigated. We found 81.4% activated lymphocytes and 5.8% plasma cells in CSF in the first week. In addition, various lymphoid features were disclosed in association: mitosis, binucleation, abnormal nuclear lobulation, Russell bodies, and morula in plasma cells. These cellular changes specifically existed in CSF of acute viral encephalitis, which were helpful to distinguish the said disease from tuberculous melitis and multiple sclerosis.
OBJECTIVE: To explore the relationship between the effects of platelet transfusion and histocompatibility antigen (HLA). METHODS: The relationship between the corrected count increment (CCI) of platelet with lymphocytotoxicity test (LCT) and human histocompatibity antigen system-I subtype (HLA-I) in 126 hospitalized patients who accepted platelet transfusion. RESULTS: Ineffective platelet transfusion was strongly related to LCT positive and LCT intensity (P < 0.01). The HLA A2 gene frequency was 0.91. The frequency was much higher than that of health human being (0.54). CONCLUSION: Positive LCT was mainly immune factor for ineffective platelet transfusion. CCI and LCT should be the routine test for platelet patients. It may improve the effects of platelet transfusion.
The effect of positive acceleration on plasma levels of calcitonin gene related peptide (CGRP), and endothelin as well as renal function in pilots were observed in this study. 20 pilots were exposed to +2.5 Gz 10 s and +3.0 Gz 10 s with an interval of 5 min without anti-G suits. Samples of plasma and serum were taken 2Omin before and after exposure. Plasma levels of CGRP and endothelin after the exposure were significantly increased (P<0.01), but alkaline phosphatase(AKP), blood levels of beta 2-microglobulin(beta 2-MG), Ca2+ in serum showed no significant change (P>0.05) as compared with those before exposure. There was a correlation between CGRP and endothlin (r=0.772, P<0.01). It is concluded that positive acceleration(+2.5, +3.0Gz) could increase plasma levels of CGRP and endothlin but did not affect renal function.
OBJECTIVES: To investigate the functions of dopamine D(1) and D(2) receptor in form deprivation myopia (FDM) and the influence of apomorphine (Apo) on inhibition of FDM in chick with radioligand binding assay (RBA). METHODS: All chicks after hatching were divided into three groups: unilateral lid suture, lid-suture and treated with Apo subconjunctival injections and normal control. After two weeks, they were refracted, then were killed, their equator dimensions of the eyeballs were measured quickly, the retinas were dissected out and pooled into three above separate groups. [(3)H]SCH 23390 and [(3)H] Spiperone were used for the saturation tests for D(1) and D(2) receptor protein respectively in the three groups. RESULTS: The numbers of both dopamine receptors in FDM increased (up-regulation). With the treatment of Apo, the numbers of both dopamine receptors decreased (down-regulation). The affinities of both receptors were of no difference. CONCLUSIONS: The dopamine receptors in chick retina are under hypersensitive state in FDM, that is related to the biological level lowering of dopamine. Apo can inhibit the development of myopia, and the D(1) and D(2) receptor numbers are decreased, showing that Apo can affect dopamine receptors in various degrees. The method of this study can provide a route to screen drugs for treatment of myopia.
The expression of the polyamine catabolic enzyme, spermidine/spermine N1-acetyltransferase (SSAT), has been associated with tumor sensitivity to antitumor polyamine analogues. In the sensitive cell types the level of SSAT is greatly induced by these agents. Although SSAT expression is regulated at many levels, the initial regulation of this X-linked gene occurs at the level of transcription. Because most previous work in human cell lines has been performed in cells of male origin and because the SSAT gene is located near the pseudoautosomal region of the X chromosome, we investigated the possibility that both copies of SSAT could be expressed in normal and tumor cells in women. DNA methyl-sensitive restriction enzyme analysis of DNA from normal peripheral lymphocytes suggested that like most X-linked genes, only one copy of SSAT is actively transcribed. However, in an examination of four representative human lung tumor cell lines derived from women, two were found to have a methylation pattern identical to male-derived cells, suggesting a reactivation of the normally inactive allele or loss of the inactive allele. Microsatellite repeat polymorphism analysis indicated that one of the lines, a female carcinoid line, NCI H727, had reactivated the previously inactive copy, thus providing H727 with two active alleles, whereas a small cell lung cancer line, H889, appears to have lost the inactive allele. Most importantly, the H727 line expressed high amounts of SSAT mRNA and protein in response to treatment with the polyamine analogue, N1,N12-bis(ethyl)spermine, a compound known to increase SSAT transcription in sensitive cell types. H727 was also the only female line that responded to treatment in a cytotoxic manner. These data suggest that both copies of the SSAT allele may be expressed and that the inappropriate expression of the second copy can lead to an increase in tumor sensitivity to polyamine analogues that induce SSAT.