Biomedical subjects
Publications and source records attributed to H Cao.
With four kinds of spectrometric analysis methods, i.e. spectrophotometric analysis, AAS, ICP-AES and IR, the interaction between Cu2+ and cholesterol in aqueous solution has been examined. The same results from the different methods showed that there was a surface adsorption between Cu2+ and cholesterol and this interaction would be prominent in physiology-like situation.
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OBJECTIVE: To construct retroviral vector carrying rapidly selective marker. METHODS: The recombination retroviral vector GCGFPPXSN was constructed by cloning the green fluorescent protein (GFP) cDNA into the retroviral vector containing putative internal ribosome entry sites GCXPXSN and transferred in ecotropic packaging cell line PE501 by electroporation method. The supernatants of the PE501GCGFPPXSN were used to infect the amphotropic packaging cell line PA317. The G418 resistant clones were selected in 4 weeks and were detectable by fluorescence microscopy or by fluorescence-activated cell sorting(FACS). RESULTS: A recombination retroviral vector GCGFPPXSN carrying rapidly selective marker GFP was constructed. GFP expression in packaging cell line PA317-GCGFPPXSN transferred by GCGFPPXSN was detected by fluorescence microscopy of FACS. PA317-GCGFPPXSN grew to a titer of 1.2 x 10(5) cpu/ml. By cocultivating retroviral vector producing cells and T lymphocytes, expression of GFP was observed in T lymphocytes 2 days after the end of the cocultivation. T lymphocytes expressing GFP were separated. CONCLUSION: The mammalian cell can be efficient gene transfected by retroviral vector carrying GFP. The use of GFP for cell marking represents an important advantage over conventional strategies which typically involve the use of neomycin resistance. GFP, in fact, allows a rapid in vitro selection of transduced cell by FACS. The selection requires only two-day culture with this retroviral vector, compared with 10-14 day culture with a classical retroviral vector.
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Positioned nucleosomes are believed to play important roles in transcriptional regulation and for the organization of chromatin in cell nuclei. Here, we have isolated the DNA segments in the mouse genome that form the most stable nucleosomes yet characterized. In separate molecules we find phased runs of three to four adenine nucleotides, extensive CA repeats, and in a few cases phased TATA tetranucleotides. The latter forms the most stable nucleosome yet characterized. One sequence with CAG repeats was also found. By fluorescence in situ hydridization the selected sequences are shown to be localized at the centromeric regions of mouse metaphase chromosomes.
The antiestrogen tamoxifen is widely used for endocrine therapy of breast cancer; however, the mechanisms of estrogen receptor-independent interactions of tamoxifen remain ill defined. Here we examine the effect of tamoxifen on the initial steps of cell signal transduction. To this end, phospholipid metabolism and protein kinase C (PKC) translocation were assessed in CCD986SK human mammary fibroblasts treated with tamoxifen. The addition of tamoxifen resulted in dose-dependent and time-dependent increases in the cellular second messengers phosphatidate (PA) and diacylglycerol (DG). On addition of ethanol to the medium, tamoxifen induced the formation of phosphatidylethanol, demonstrating that tamoxifen activates phospholipase D (PLD). Cellular DG also increased in the presence of ethanol, showing that tamoxifen also activates phospholipase C (PLC). In cells prelabeled with choline and ethanolamine, tamoxifen caused increases in choline, phosphorylcholine, ethanolamine and phosphorylethanolamine. Structure-activity relationship studies for activation of PLD revealed that tamoxifen was the most effective, whereas 4-hydroxy tamoxifen was nearly devoid of activity. Phorbol diesters also activated PLD, but estrogen had no influence. Pretreatment of cells with phorbol dibutyrate (PKC down-regulation protocol) blocked phorbol diester- and tamoxifen-induced PLD activity. Exposure of cells to the PKC inhibitor GF 109203X diminished tamoxifen-induced PLD activity. Addition of tamoxifen to cultures elicited selective membrane association of PKC epsilon. We conclude that tamoxifen exerts considerable extra-nuclear influence at the transmembrane signaling level. These events may contribute to effects beyond the scope of estrogen receptor-dependent actions.
The Arabidopsis NPR1 gene controls the onset of systemic acquired resistance (SAR), a plant immunity, to a broad spectrum of pathogens that is normally established after a primary exposure to avirulent pathogens. Mutants with defects in NPR1 fail to respond to various SAR-inducing treatments, displaying little expression of pathogenesis-related (PR) genes and exhibiting increased susceptibility to infections. NPR1 was cloned using a map-based approach and was found to encode a novel protein containing ankyrin repeats. The lesion in one npr1 mutant allele disrupted the ankyrin consensus sequence, suggesting that these repeats are important for NPR1 function. Furthermore, transformation of the cloned wild-type NPR1 gene into npr1 mutants not only complemented the mutations, restoring the responsiveness to SAR induction with respect to PR-gene expression and resistance to infections, but also rendered the transgenic plants more resistant to infection by P. syringae in the absence of SAR induction.
BACKGROUND: RPE transplantation offers the possibility of treating certain forms of retinal degeneration. Understanding how to optimize the surgical technique for performing RPE transplantation, especially in primates, is therefore of considerable interest. METHODS: Fifteen patch RPE transplants were performed in six monkeys. The transplant sites were examined at follow-up by ophthalmoscopy, biomicroscopy, fluorescein angiography and histology. Foveal and peripheral retinal transplants were compared. RESULTS: Human fetal RPE xenografts can survive without rejection for at least 6 months after transplantation in monkey retina. Such grafts form a basal lamina and make intimate contacts with the outer segments of the host. Both rods and cones retain a normal appearance when in contact with unrejected transplants. Rejection occurred in only 30% (3/10) of the peripheral but in 60% (3/5) of the foveal transplants. CONCLUSIONS: Cultured human fetal RPE patch transplants can survive and maintain local photoreceptor integrity for relatively long periods of time in monkey subretinal space without immunosuppression. Rejection, when it occurs, is more frequent near the fovea.
Despite recent advances in antiviral therapy for human immunodeficiency virus (HIV) infection, successful global intervention will require an effective vaccine. Expanding evidence suggests that cytotoxic T-lymphocyte (CTL) responses will be an important component of such a vaccine. The varying geographic distribution of HIV type 1 (HIV-1) clades, with the relative absence of clade B HIV-1 outside the developed world, is considered a major obstacle to the development of a single efficacious vaccine. An understanding of cross-reactive CTL responses between different HIV-1 clades is crucial in the design of a vaccine which will be broadly immunogenic. In this study, we examined the ability of HIV-1 Gag-, reverse transcriptase-, and Env-specific CTL clones isolated from individuals infected in the United States to recognize non-B clade viral sequences and found that all were cross-reactive with the majority of non-B clade viral sequences tested. We next studied HIV-1-specific CTL responses in African individuals infected with clade A, C, or G virus and evaluated cross-recognition of clade B virus. Of 14 persons evaluated, all demonstrated cross-reactivity with the U.S. clade B viral constructs. We conclude that significant CTL cross-reactivity exists between clade B and non-B epitopes, suggesting that CTL cross-recognition among HIV-1 clades is more widespread than anticipated and that a vaccine based on a single clade may be broadly applicable.
Although CD8+ lymphocytes in human immunodeficiency virus type 1 (HIV-1)-infected individuals have been demonstrated to suppress viral replication, the mechanisms of inhibition have not been defined precisely. A large body of evidence indicates that these cells act via soluble inhibitory factors, but the potential role of HLA class I-restricted cytolysis has remained controversial. Here we demonstrate that HIV-1-specific cytotoxic T lymphocytes (CTL) mediate antiviral suppression by both cytolytic and noncytolytic mechanisms. The predominant mechanism requires direct contact of CTL with the infected cells, is HLA class I restricted, and can achieve complete elimination of detectable virus in infected cell cultures. Inhibition occurs even at high multiplicities of infection or at ratios of CTL to CD4 cells as low as 1:1,000. The other mechanism is mediated by soluble inhibitory factors which are triggered in an antigen-specific and HLA-restricted fashion but then act without HLA restriction. These include MIP-1alpha, MIP-1beta, and RANTES, as well as a distinct factor(s) capable of inhibiting HIV-1 strains insensitive to these chemokines. These data indicate that HIV-1-specific CTL are potent mediators of HIV-1 suppression at cell ratios existing in vivo and demonstrate an antigen-specific trigger for CD8+ cell-derived soluble inhibitory factors. These results suggest that CTL play an important role in the observed antiviral activity of CD8+ cells from infected individuals.
The bona fide 5S rRNA genes in the rat are found in a 1.8-kb tandem repeat and the pseudogenes occur in a 2.5-kb tandem repeat. Three bona fide 5S rRNA genes and one gene variant with one base substitution in the coding region were isolated from the 1.8-kb repeat. Six pseudogenes were isolated from the 2.5-kb repeat. The total number of genes/gene variants/pseudogenes is 700-1200 copies per haploid genome, and the pseudogene repeat contains about 50% more 5S rDNA related sequences compared with the bona fide gene repeat. Various well-defined 5' - and 3'-flanking sequences of the bona fide gene and of the pseudogene were used for in situ hybridization to metaphase chromosomes. The results showed that the bona fide 5S rRNA gene repeat Rn5s maps to chromosome 19q12 and the pseudogene repeat Rn5sp maps to 12q12.
DNA fingerprinting for Taraxacum mongolicum and its adulterants of six species of Compositae was demonstrated with random-primed polymerase chain reaction (PCR) including arbitrarily-primed PCR (AP-PCR) and random-amplified polymorphic DNA(RAPD). Distinctive genomic fingerprints from DNA of Taraxacum mongolicum and its adulterants were generated with two long (20-24 mer) and one short (10 mer) random-chosen primers Taraxacum mongolicum can be distinguished from six adulterants according to the banding patterns of their amplified DNA on agarose gels. Moreover, that Taraxacum mongolicum and its adulterant are unrelated. The Similarity Indexes values of the genomic fingerprints. Results showed AP-PCR and RAPD might be used for identifying Chinese drugs.
OBJECTIVE: To obtain recombinant human thrombopoietin (rhTpo). METHODS: rhTpo gene was cloned from fetal liver mRNA by means of RT-PCR and subcloned into permanent expressing plasmid RC/CMV in order to form a recombinant permanent expressing plasmid RC/CMV/Tpo. RESULTS AND CONCLUSION: After being transferred into CHO cells, the eukaryotic permanent expression of rhTpo, which can enhance murine megakaryocyte colony growth in vitro, was obtained.
OBJECTIVE: To explore an approach to the reduction of activation induced apoptosis of T cell so as to maximize the number of CTL and enhance the tumor cytotoxicity. METHODS: CD3-induced Jurkat cell line was used as the activated T cell apoptosis model,and the antisense Fas cDNA was introduced into Jurkat cells with retroviral vector. RESULTS: The expression level of Fas protein in Jurkat cells transducted with the retroviral vector was decreased. Apoptosis was reduced in antisense Fas-transfected Jurkat cells after anti-CD3 or anti-Fas treatment. CONCLUSION: Antisense blocking of Fas expression can partially inhibit Jurkat cell apoptosis induced by anti-CD3 or anti Fas.
OBJECTIVE: To investigate the perinatal infection of human parvovirus B19 in pregnant women and their fetuses. METHODS: 350 sera of normal pregnant women and 9 sera of abnormal fetuses were investigated by polymerase chain reaction (PCR) to check the parvovirus B19 DNA. RESULTS: The prevalence of parvovirus B19 DNA was 1.14% and 0.28% in the sera of normal pregnant women and umbilical cords, respectively. Parvovirus B19 DNA was detected in 6 sera of pregnant women and umbilical cords from 9 abnormal fetuses parvovirus DNA could be detected by in situ hybridization in the nuclei of immature hematopoetic cells within fetal brain or spleen whose PCR tests were positive. CONCLUSION: The results suggest that parvovirus B19 infection do exist in pregnant women and their neonates as well as fetuses in our country. PCR and in situ hybridization could provide more sensitive, more accurate and specific methods for clinical pathologic diagnosis and epidemic investigation.
OBJECTIVE: To observe the effect of theo-polyphenols (TP) on oxygen free radicals and hemorrheology in patients with essential hypertension. METHODS: Hemorheology as well as oxygen free radical of 38 patients were determined before and after TP treatment. RESULTS: TP markedly increased RBC-SOD activity, reduced the level of serum MDA, the whole blood viscosity, plasma viscosity and the level of plasma fibrinogen. The efficacy of TP was higher than that of aspirin. CONCLUSION: The TP had remarkable effects of anti-oxygen free radicals, and could improve hemorheology.
A novel cDNA, TR2L, isolated from murine NIH 3T3 fibroblasts, was found to modulate tumor necrosis factor (TNF)-mediated apoptosis in murine L929 fibrosarcoma cells. The full-length cDNA (853 bp) encodes a predicted coding region of 56 amino acids (6.3 kD), with 53.6% identity to the C-terminus of rat transcriptional activator FE65. When expressed stably in L929 cells, TR2L protein inhibited TNF cytotoxic response. In contrast, TR2L enhanced anti-Fas antibodies/actinomycin D (ActD)-mediated L929 apoptosis. Alteration of TR2L function occurred by tagging this protein with a 6xHis fragment to the N-terminus (designated 6xH-TR2L). L929 cells which stably expressed 6xH-TR2L acquired a significantly enhanced TNF apoptotic response and increased genomic DNA fragmentation compared to control cells. Enhanced cell death also occurred in these 6xH-TR2L-expressing cells under serum starvation conditions. In contrast, the anti-Fas/ActD-mediated apoptosis was blocked by the 6xH-TR2L protein. Functional role of TR2L protein in regulation of cancer cell susceptibility to TNF-and Fas ligand-mediated apoptosis is suggested.