Biomedical subjects

Z Cai

Publications and source records attributed to Z Cai.

At least 73 records · Page 4Linked to original sources

PubMed2000

Regulation of transporter associated with antigen processing by phosphorylation.

The ATP-binding cassette transporter associated with antigen processing (TAP) is required for transport of antigenic peptides, generated by proteasome complexes in the cytoplasm, into the lumen of the endoplasmic reticulum where assembly with major histocompatibility complex class I molecules takes place. The TAP transporter is a heterodimer of TAP1 and TAP2. Here we show that both TAP1 and TAP2 are phosphorylated under physiological conditions. Phosphorylation induces formation of high molecular weight TAP complexes that contain TAP1, TAP2, tapasin, and class I heterodimers. In addition, a 43-kDa phosphoprotein, which appears to be a kinase, is contained in the phosphorylated TAP-containing complexes. Phosphorylated TAP complexes are able to bind peptides and ATP, however, they are not capable of transporting peptides. After de-phosphorylation, TAP complexes regain the ability to transport peptides. Interestingly, phosphorylation levels of TAP complexes induced by viral infection inversely correlates with a significant reduction in TAP-dependent peptide transport activity. Enhanced TAP phosphorylation appears to be one of several strategies that viruses have exploited to better escape from host immune surveillance. These results demonstrate that major histocompatibility complex class I antigen processing and presentation is modulated by reversible TAP phosphorylation, and implicate the importance of TAP phosphorylation in the regulation of cytotoxic immune response.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2000

cAMP-dependent protein kinase inhibits mGluR2 coupling to G-proteins by direct receptor phosphorylation.

One of the primary physiological roles of group II and group III metabotropic glutamate receptors (mGluRs) is to presynaptically reduce synaptic transmission at glutamatergic synapses. Interestingly, previous studies suggest that presynaptic mGluRs are tightly regulated by protein kinases. cAMP analogs and the adenylyl cyclase activator forskolin inhibit the function of presynaptic group II mGluRs in area CA3 of the hippocampus. We now report that forskolin has a similar inhibitory effect on putative mGluR2-mediated responses at the medial perforant path synapse and that this effect of forskolin is blocked by a selective inhibitor of cAMP-dependent protein kinase (PKA). A series of biochemical and molecular studies was used to determine the precise mechanism by which PKA inhibits mGluR2 function. Our studies reveal that PKA directly phosphorylates mGluR2 at a single serine residue (Ser(843)) on the C-terminal tail region of the receptor. Site-directed mutagenesis combined with biochemical measures of mGluR2 function reveal that phosphorylation of this site inhibits coupling of mGluR2 from GTP-binding proteins

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed2000

Reduced nitric oxide is involved in prenatal ischemia-induced tolerance to neonatal hypoxic-ischemic brain injury in rats.

To explore the role of nitric oxide (NO) in the hypoxic-ischemic (HI) tolerance phenomenon, NO production and brain injury following neonatal hypoxia-ischemia (induced by unilateral common carotid artery ligation followed by hypoxic exposure) were assessed in rat pups with or without HI preconditioning. A previously demonstrated prenatal HI rat model of preconditioning was used in this study. On G17, rat fetuses were subjected to either HI in utero (PreHI) for 30 min or a sham operation (SH). The PreHI treatment provided significant protection against neonatal HI-induced brain injury, as indicated by decreased ipsilateral brain weight reduction, less severe tissue damage, and decreased activation of caspase-3. Concomitant with the protective effect of prenatal HI preconditioning, elevation of nitrite/nitrate content in the ipsilateral cortex of the brain, as an indirect measure of NO production, was significantly lower in the PreHI group than in the SH group following neonatal HI. The protective effect of prenatal HI preconditioning could be reversed by sodium nitroprusside (SNP), a spontaneous NO donor, while SNP had no effect on neonatal HI-induced brain injury in the SH group. Intraperitoneal administration of SNP to pups from the PreHI group (2 mg/kg, 24 and 1.5 h before neonatal HI) increased neonatal HI-induced brain injury similar to that observed in the SH group. On the other hand, L-N(G)-nitro-arginine (2 mg/kg, i.p., 1.5 h before the hypoxic exposure), an NO synthase inhibitor, significantly attenuated neonatal HI-induced brain injury in the SH group. The overall results indicate that reduced NO production in the preconditioned rat brain contributes to prenatal HI-induced tolerance to neonatal HI brain injury.

Animals↗

PubMed2000

Two mechanisms of genistein inhibition of cystic fibrosis transmembrane conductance regulator Cl- channels expressed in murine cell line.

1. The isoflavone genistein may either stimulate or inhibit cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels. To investigate how genistein inhibits CFTR, we studied CFTR Cl- channels in excised inside-out membrane patches from cells expressing wild-type human CFTR. 2. Addition of genistein (100 microM) to the intracellular solution caused a small decrease in single-channel current amplitude (i), but a large reduction in open probability (Po). 3. Single-channel analysis of channel block suggested that genistein (100 microM) may inhibit CFTR by two mechanisms: first, it may slow the rate of channel opening and second, it may block open channels. 4. Acidification of the intracellular solution relieved channel block, suggesting that the anionic form of genistein may inhibit CFTR. 5. Genistein inhibition of CFTR Cl- currents was weakly voltage dependent and unaffected by changes in the extracellular Cl- concentration. 6. Channel block was relieved by pyrophosphate (5 mM) and ATP (5 mM), two agents that interact with the nucleotide-binding domains (NBDs) of CFTR to greatly stimulate channel activity. 7. ATP (5 mM) prevented the genistein-induced decrease in Po, but was without effect on the genistein-induced decrease in i. 8. The genistein-induced decrease in i was voltage dependent, whereas the genistein-induced decrease in Po was voltage independent. 9. The data suggest that genistein may inhibit CFTR by two mechanisms. First, it may interact with NBD1 to potently inhibit channel opening. Second, it may bind within the CFTR pore to weakly block Cl- permeation.

ATP-Binding Cassette Transporters↗

PubMed2000

T cells can use either T cell receptor or CD28 receptors to absorb and internalize cell surface molecules derived from antigen-presenting cells.

At the site of contact between T cells and antigen-presenting cells (APCs), T cell receptor (TCR)-peptide-major histocompatibility complex (MHC) interaction is intensified by interactions between other molecules, notably by CD28 and lymphocyte function-associated antigen 1 (LFA-1) on T cells interacting with B7 (B7-1 and B7-2), and intracellular adhesion molecule 1 (ICAM-1), respectively, on APCs. Here, we show that during T cell-APC interaction, T cells rapidly absorb various molecules from APCs onto the cell membrane and then internalize these molecules. This process is dictated by at least two receptors on T cells, namely CD28 and TCR molecules. The biological significance of T cell uptake of molecules from APCs is unclear. One possibility is that this process may allow activated T cells to move freely from one APC to another and eventually gain entry into the circulation.

Animals↗

PubMed2000

Adenovirus-mediated wild-type-p53-gene expression sensitizes TNF-resistant tumor cells to TNF-induced cytotoxicity by altering the cellular redox state.

We have shown that the loss of p53 function contributed to resistance of tumor cells to TNF-induced cytotoxicity. In the present study, we evaluated the effect of wild-type p53 (wt-p53) expression on TNF sensitivity, by introducing wt-p53 into MCF7/Adr cells in which p53 was deleted, via a recombinant adenovirus encoding p53 (Ad-p53). Our results indicate that infection with Ad-p53 (50-100 viral particles per cell) resulted in pronounced cytotoxicity, whereas infection with 10 viral particles per cell, which was weakly toxic for the MCF7/Adr cells, sensitized these cells to TNF-induced cell death. Moreover, expression of wt-p53 in MCF7/Adr cells induced the production of reactive oxygen intermediates (ROIs) and caused glutathione (GSH) depletion, indicating disturbances in the cellular redox state. Additional treatment of cells with the anti-oxidant and glutathione (GSH) precursor N-acetylcysteine (NAC) resulted in inhibition of p53-induced ROIs production and in partial restoration of intracellular GSH levels, which was associated with the ability of NAC to inhibit p53-modulated TNF-induced cytotoxicity. Interestingly, Ad-p53 was able to inhibit TNF-induced MnSOD mRNA expression in MCF7/Adr cells, which might contribute to the sensitization of cells to the cytotoxic action of TNF. Taken together, our data strongly suggest that wt-p53 expression sensitizes TNF-resistant MCF7 cells with p53 deletion to TNF-induced cell death by a pathway that is dependent on ROIs production.

Acetylcysteine↗

PubMed2000

Metabotropic glutamate receptors in acutely isolated hippocampal astrocytes: developmental changes of mGluR5 mRNA and functional expression.

We previously found that 82% of glial fibrillary acidic protein (GFAP)-positive hippocampal astrocytes acutely isolated from P1-10 rats responded to glutamate (Glu) with transient intracellular calcium increases via activation of a Group I metabotropic glutamate receptor (mGluR). Fewer cells responded to ATP and none to serotonin (5-HT). In this study we asked the question whether hippocampal astrocytes in older animals retain this relative pattern of expression. We have found that 77% of GFAP (+) cells from P11-20 rats responded to 50 microM Glu, 43% to ATP, and none to 5-HT. Thirty-three percent of GFAP (+) cells from P25-35 rats responded to Glu, 12% to ATP and 3% to 5-HT. In the case of the responses to Glu, pharmacological characterization and single-cell RT-PCR data confirmed that these responses were mediated by the mGluR5 subtype of group I mGluRs. Also, fewer (36%) GFAP mRNA (+) cells from P25-35 rats expressed detectable mGluR5 mRNA than those from P11-20 rats (77%). This number essentially corresponds to the number of GFAP(+) showing a Ca(2+) response to Glu. Both mGluR5a and b were expressed with equal frequency in cells from P11-20 rats, but the b form predominated in cells from older animals. Overall, our studies show that expression of mGluR5 in hippocampal astrocytes decreases with increasing age and the "a" splice variant declines to a greater extent than the "b" splice variant, corresponding to the developmental changes shown in total tissue for mGluR5.

Aging↗

PubMed2000

Simultaneous quantitative cassette analysis of drugs and detection of their metabolites by high performance liquid chromatography/ion trap mass spectrometry.

A method using high performance liquid chromatography (HPLC) coupled with ion trap mass spectrometry (MS) for simultaneous quantification of multiple drugs and detection of their metabolites is described. The new approach offers a significant increase in analytical throughput and is illustrated with analysis of the in vitro metabolism of 19 alpha-1a receptor antagonists. The compounds were separated into four cassette groups by using a computer program as well as by manual examination. The samples from incubation with dog liver microsomes were pooled into the designed cassette groups and analyzed by HPLC/electrospray (ESI) ion trap MS in full-scan mode. The metabolic stability of the drugs was determined by comparing their signals after incubation for 0 and 60 min, respectively. The quantitative results from the cassette analysis procedure agreed well with those obtained from conventional discrete analysis. In addition, the technique allowed simultaneous detection of metabolites formed during the same incubation without having to reanalyze the samples. The metabolites were first characterized by nominal mass measurement of the corresponding protonated molecules. Subsequent multi-stage tandem mass spectrometry (MS(n)) on the ion trap instrument allowed confirmation of the detected metabolites.

Adrenergic alpha-Antagonists↗

PubMed2000

Acutely isolated astrocytes as models to probe astrocyte functions.

Neuroscientists have become increasingly aware and accepting of the concept that astrocytes likely have many important functions in the CNS. One limitation in establishing these functions is the usual problem of what constitutes suitable experimental approaches. A major experimental step for functional studies of astrocytes has been the widespread use of primary astrocyte cultures, an approach that Leif Hertz pioneered. However, it is now becoming clear that, building on this work, an experimental paradigm shift is now needed. Namely, to increasingly study preparations corresponding to in situ conditions, such as slices. An alternative experimental system where the cells have some of the technical advantages of primary astrocyte cultures is freshly isolated astrocytes. Recent experiments from our laboratory have shown metabotropic glutamate receptor expression by such cells. Examples are given of how functional receptor studies and channel activity measured by patch clamp electrophysiology can be combined with single cell RT-PCR to define further the receptor or channel type are described to illustrate the uses of such preparations.

Animals↗

PubMed2000

Haploinsufficiency of AML1 affects the temporal and spatial generation of hematopoietic stem cells in the mouse embryo.

The AML1:CBFbeta transcription factor complex is essential for definitive hematopoiesis. Null mutations in mouse AML1 result in midgestational lethality with a complete lack of fetal liver hematopoiesis. While the cell autonomous nature and expression pattern of AML1 suggest an intrinsic role for this transcription factor in the developing hematopoietic system, no direct link to a functional cell type has been made. Here, we examine the consequences of AML1 loss in hematopoietic stem cells (HSC) of the mouse embryo. We demonstrate an absolute requirement for AML1 in functional HSCs. Moreover, haploinsufficiency results in a dramatic change in the temporal and spatial distribution of HSCs, leading to their early appearance in the normal position in the aorta-gonad-mesonephros region and also in the yolk sac.

Animals↗

PubMed2000

Shear stress-mediated NO production in inner medullary collecting duct cells.

Recent evidence suggests that nitric oxide (NO) within the inner medullary collecting duct (IMCD) functions to regulate sodium and water reabsorption. Because fluid shear stress has been shown to increase NO production in endothelial and vascular smooth muscle cells, experiments were designed to determine whether a similar mechanism exists in IMCD cells. Cultured IMCD-3 cells derived from murine IMCD were subjected to 60 min of pulsatile shear stress. Nitrite production (2,3-diaminonaphthalene fluorometric assay) increased 12-, 16-, and 23-fold at 3.3, 10, and 30 dyn/cm(2), respectively, compared with static control cultures. Preincubation with the non-isoform-specific NO synthase inhibitor nitro-L-arginine methyl ester reduced nitrite production by 83% in response to 30 dyn/cm(2). Western blotting and immunofluorescence analysis of static IMCD-3 cell cultures revealed the expression of all three NO synthase isoforms (NOS-1 or neuronal NOS, NOS-2 or inducible NOS, and NOS-3 or endothelial NOS) in IMCD-3 cultures. These results indicate that NO production is modulated by shear stress in IMCD-3 cells and that fluid shear stress within the renal tubular system may play a role in the regulation of sodium and water excretion by control of NO production in the IMCD.

Animals↗

PubMed2000

Cytokine induction in fetal rat brains and brain injury in neonatal rats after maternal lipopolysaccharide administration.

Induction of proinflammatory cytokines has been proposed to be a link between prenatal maternal intrauterine infection and neonatal brain damage. It is known that the endotoxin, lipopolysaccharide (LPS), released during bacterial infection crosses the placenta. Cytokine induction in the fetal rat brain after maternal administration of LPS was determined by reverse transcriptase-polymerase chain reaction method. LPS suspension in pyrogen-free saline was administered (i.p.) to pregnant rats at 18 d of gestation. The control group was treated with pyrogen-free saline. Expression of the proinflammatory cytokines, tumor necrosis factor-alpha and IL-1beta mRNA, in the fetal rat brain was increased in a dose-dependent manner at 1 h after LPS administration. The great increase in expression of IL-1beta mRNA was only observed at 1 h after injection of LPS (4 mg/kg), whereas the increased expression of tumor necrosis factor-alpha was still detectable from 4 to 24 h after LPS administration. Brain injuries were examined by immunohistochemistry in 8-d-old rat pups born to the dams that were consecutively treated with LPS (500 microg/kg) or pyrogen-free saline on gestation d 18 and 19. No apparent necrotic tissue damage was found in either the LPS group or the control group. Myelin basic protein staining, as a marker of myelin, was clearly observed in the internal capsule and the fimbria hippocampus in the rat brain from the control group. Myelin basic protein staining was much less and weaker in the brains of the LPS-treated group. Glial fibrillary acidic protein-positive astrocytes were observed in both the control and the LPS-treated groups. The LPS-treated group appeared to have more glial fibrillary acidic protein-positive astrocytes in the hippocampal and the cortex areas of the brain than the control group. Immunoblotting data showed that glial fibrillary acidic protein content in the cortex or the hippocampus of the LPS-treated rat brain was higher than in the control group. OX-42-positive staining (a marker of the type 3 complement receptors) of microglial cells was greatly reduced in the 8-d-old rat brain after maternal LPS administration. However, histochemistry with tomato lectin showed that staining of both amoeboid and ramified microglial cells in the LPS-treated rat brain was similar to that in the control group. The overall results indicate that maternal LPS administration induces an increased expression of IL-1beta and tumor necrosis factor-alpha mRNA in the fetal brain. Maternal LPS administration also increases glial fibrillary acidic protein-positive astrocytes, decreases myelin basic protein and alters immunoreactivity of microglia in the brain of offspring. Although results from the current study do not provide direct evidence linking LPS-induced cytokines with the abnormalities in the neonatal rat brain, our animal model may be appropriate for exploring the mechanisms involved in the effects of maternal infection on glial cells in the brains of offspring.

Animals↗

PubMed2000

An experimental study on the developmental malformations of the trisomy 18 NMRI mice.

OBJECTIVE: To study the incidence of trisomy 18 in NMRI mice and its developmental malformations. METHODS: Sixty female NMRI mice and 11 male mice with the Rb(2.18) 6 Rma and Rb (1 +/-18) 10 Rma chromosomes were used. On the gestational days 16 and 17, the pregnant mice were randomly killed by cervical dislocation. A total of 823 implants were gained from 60 litters; they comprised 569 living foetuses, and chromosomal examination was performed in the generated living foetuses. Gross morphological study was performed in all living fotuses. RESULTS: Ninety-five foetuses were identified as trisomic foetuses in all of 569 living foetuses. Among them, there were 68 trisomy 18 NMRI mice with cleft palate. Gross morphological study showed that trisomy 18 foetuses had significant body flexion. Some cases of trisomy 18 showed shortened limbs, oedematous body or exencephaly as additional symptoms. CONCLUSION: Developmental malformations in trisomy 18 NMRI mice showed a seried of abnormalities; they had not only the underdeveloped body weight, but also the highter incidence of congenital cleft palate.

Animals↗

PubMed2000

The expression kinetics of myogenin in facial muscle denervation.

OBJECTIVE: To study the expression kinetics of myogenin in long-term denervated facial muscle and to explore the possibility of gene therapy for facial muscle paralysis with myogenin gene. MATERIALS AND METHODS: In 48 New Zealand rabbits, buccal muscle paralysis of one side was produced by excision of 1 cm segment buccal branch of the facial nerve. The opposite side served as controls. The animals were sacrificed and the buccal muscles of both sides were removed for examination at 1 day, 3 days, 1 week, 2 weeks, 1 month, 2 months, 4 months, and 6 months after the initial operation. The myogenin expression in denervated and innervated buccal muscles was analyzed by Western blot. Satellite cell proliferation was detected with proliferating cell nuclear antigen (PCNA) analysis. Muscle nucleic acid concentration was determined through acridine orange (AO) staining method. RESULTS: Myogenin expression increased to the highest level at 3 days after denervation, thereafter it gradually decreased. The Western blotting signal for myogenin intensified again after 1 month and at 4 months. In contrast, the highest expression of the controls (innervated muscles) were observed at 1 month. These changes in myogenin expression in denervated buccal muscles were consistent with the change in satellite cell proliferation and in muscle nucleic acid concentration. CONCLUSION: Myogenin protein expression in longterm facial muscle denervation is closely associated with satellite cell regeneration. Myogenin may promote satellite cell differentiation, and therefore may improve the treatment of facial paralysis.

Acridine Orange↗

PubMed2000

[Study of human tissue engineering cartilage].

OBJECTIVE: To investigate the technique of human tissue-engineered cartilage and to study the medical collagen membrane of guided tissue regeneration (GTR) as the carrier of in vitro chondrocytes culture. METHODS: Tissue engineering technique was used to make human Tissue-engineered cartilage, which was examined histologically and functionally. RESULTS: It was found that the chondrocytes seeded on the medical collagen membrane of GTR grew well. A layer of milk white and cartilage-like tissue grew on the surface of medical collagen membrane of GTR after 1 week. It was demonstrated that the cartilage-like tissue was strong enough to be transferred after being implanted for 8 weeks. The cartilage-like tissue was proved to be human tissue engineered cartilage by HE stain and Alcian blue-poncean S stain. The chondrocytes could secrete chondroitin sulfate as proved by Lev-Spicer stain. CONCLUSIONS: The medical collagen membrane of GTR has characteristics of three-dimensional structure and cell reticular function, and it has the possibility to be developed as a natural scaffold for tissue engineering. The results indicate that it is possible to make human tissue-engineered cartilage with tissue-engineering technique.

Animals↗

PubMed2000

[Effect of land management in winter crop season on seasonal variations of CH4 emissions from rice paddy soils].

A pot experiment in greenhouse was carried out to study seasonal variations of CH4 fluxes from rice paddy soils and the effect of land management in winter crop season on the seasonal variations. The results showed that four emission peaks occurred respectively 13 days after flooding and 7, 40, 91 days after rice transplanting, and CH4 emission amounts during the first 30 days after flooding accounted for as high as 67.5%, 35.5% and 33.3% of the total emission amounts during the observing period of 133 days for treatments with dry fallow but rice straw being applied just before flooding, alfalfa, and flooded fallow in winter crop season. However, for treatments with dry fallow but rice straw being applied before winter crop cultivating and winter wheat in winter crop season, CH4 emission amounts during the first 50 days after flooding just accounted for 0.27% and 6.74 of the total emission amounts during the observing period, and CH4 fluxes thereafter were also very small. Difference in the seasonal variation patterns of soil Eh due to land management in winter crop season was the main reason why seasonal variation patterns of CH4 fluxes from different treatments were different.

Methane↗

PubMed2000

[Morphometrical study on the development of the trisomy-18 with cleft palate and euploid mice].

OBJECTIVE: To quantitatively compare the whole body, the lateral maxillofacial profile in Ts18 foetuses with cleft palate (Ts + CP) and euploid foetuses in order to answer the question whether this animal model is associated with maldevelopment or hypoplasia of the craniomaxillofacial region. METHODS: Sixty female Hanmice and 11 male mice with the Rb(2.18) 6Rma and Rb(1.18) 10Rma chromosomes were used to generate 569 living foetuses. Of these, 52 pairs Ts + CP and euploid foetuses were selected according to about equal body weight, and used in gross morphometric study. RESULTS: Ts + 18 foetuses had greater body flexion, a significant maxillary and mandibular retrusion, also mandible in Ts + CP foetuses were smaller than in euploids without cleft. CONCLUSIONS: This animal model of congenital cleft palate developed hypoplasia of the maxilla and mandible.

Animals↗

PubMed2000

[Gil-Vernet anti-reflux operation by united application of television laparoscopy and vesicourethral laparoscopy].

OBJECTIVE: To discuss the possibility of united application of television laparoscopy and vesicourethral laparoscopy in Gil-Vernet operation. METHODS: The animal model of vesicourethral reflux (VUR) was established using rabbits. The Gil-Vernet operation was carried out by united application of television laparoscopy and vesicourethral laparoscopy. RESULTS: The VUR model was successfully established in 13 rabbits, and the reflux was successfully cured by microsurgical methods. CONCLUSIONS: This method had the advantages of television laparoscopy, vesicourethral laparoscopy and the Gil-Vernet such as no dissection of inner part of ureter-bladder wall, small wound, less bleeding, fast recovery, less complication and possible repetitive operation. It can be used in ill children with VUR.

Animals↗