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Biomedical subjects

D Y Zhao

Publications and source records attributed to D Y Zhao.

At least 19 recordsLinked to original sources
PubMed2001

Identification and quantitation of carotenoids and their metabolites in the tissues of the human eye.

There is increasing evidence that the macular pigment carotenoids, lutein and zeaxanthin, may play an important role in the prevention of age-related macular degeneration, cataract, and other blinding disorders. Although it is well known that the retina and lens are enriched in these carotenoids, relatively little is known about carotenoid levels in the uveal tract and in other ocular tissues. Also, the oxidative metabolism and physiological functions of the ocular carotenoids are not fully understood. Thus, we have set out to identify and quantify the complete spectrum of dietary carotenoids and their oxidative metabolites in a systematic manner in all tissues of the human eye in order to gain better insight into their ocular physiology. Human donor eyes were dissected, and carotenoid extracts from ocular tissues [retinal pigment epithelium/choroid (RPE/choroid), macula, peripheral retina, ciliary body, iris, lens, vitreous, cornea, and sclera] were analysed by high-performance liquid chromatography (HPLC). Carotenoids were identified and quantified by comparing their chromatographic and spectral profiles with those of authentic standards. Nearly all ocular structures examined with the exception of vitreous, cornea, and sclera had quantifiable levels of dietary (3R,3'R,6'R)-lutein, zeaxanthin, their geometrical (E / Z) isomers, as well as their metabolites, (3R,3'S,6'R)-lutein (3'-epilutein) and 3-hydroxy-beta,epsilon-caroten-3'-one. In addition, human ciliary body revealed the presence of monohydroxycarotenoids and hydrocarbon carotenoids, while only the latter group was detected in human RPE/choroid. Uveal structures (iris, ciliary body, and RPE/choroid) account for approximately 50% of the eye's total carotenoids and approximately 30% of the lutein and zeaxanthin. In the iris, these pigments are likely to play a role in filtering out phototoxic short-wavelength visible light, while they are more likely to act as antioxidants in the ciliary body. Both mechanisms, light screening and antioxidant, may be operative in the RPE/choroid in addition to a possible function of this tissue in the transport of dihydroxycarotenoids from the circulating blood to the retina. This report lends further support for the critical role of lutein, zeaxanthin, and other ocular carotenoids in protecting the eye from light-induced oxidative damage and aging.

Carotenoids↗
PubMed2000

Direct imaging of the pores and cages of three-dimensional mesoporous materials.

Mesostructured composite materials, with features ranging from 20 to 500 A in size, are obtained by the kinetically controlled competitive assembly of organic and inorganic species into nanostructured domains. Short-range order is limited, and long-range order is determined by weak forces such as van der Waals or hydrogen-bonding. Three-dimensional mesoporous materials obtained by removing the organic phase are of particular interest for applications such as catalysis and chemical sensing or separation, for which structural features such as cavity shape, connectivity and ordered bimodal porosity are critical. But atomic-scale structural characterization by the usual diffraction techniques is challenging for these partially ordered materials because of the difficulty in obtaining large (> 10 microm) single crystals, and because large repeat spacings cause diffraction intensities to fall off rapidly with scattering angle so that only limited small-angle data are available. Here we present a general approach for the direct determination of three-dimensional mesoporous structures by electron microscopy. The structure solutions are obtained uniquely without pre-assumed models or parametrization. We report high-resolution details of cage and pore structures of periodically ordered mesoporous materials, which reveal a highly ordered dual micro- and mesoscale pore structure.

Journal Article↗
PubMed2000

Brain distribution and efficacy of carbamazepine in kainic acid induced seizure in rats.

To investigate the relationships between carbamazepine (CBZ) concentrations in serum and the brain, and the anticonvulsive efficacy in kainic acid (KA) induced seizures in rats, adult Wistar rats (n=25) were intraperitoneally given 40 mg/kg CBZ, followed by 15 mg/kg KA (n=20) or saline (control, n=5). At 90 min after the injection, CBZ concentrations in 5 rats without seizures (CBZ effective group), 5 rats with seizures (CBZ no-effective group) and five control rats were measured. Serum and brain tissues from six areas (cortex, brain stem, cerebellum, thalamus, hippocampus and striatum) were used for CBZ assay. CBZ was measured using a EMIT immunoassay kit. In blood, CBZ was higher in rats treated with CBZ+KA than in a control group (CBZ+saline). In the brain, the effective group demonstrated significantly high CBZ concentration in the hippocampus. KA appeared to raise serum CBZ level when it was given in combination with CBZ. This was probably caused by the accelerated absorption of CBZ from local site as the results of an increased metabolic rate and the more demands for blood supply after KA treatment. The positive correlation between efficacy of CBZ and the concentration in the hippocampus suggests that CBZ levels in the hippocampus is closely correlated with the efficacy of CBZ against KA induced seizures.

Animals↗
PubMed2000

Anterior optic nerve microvascular changes in human glaucomatous optic neuropathy.

The microvascular changes in the anterior optic nerve in human glaucomatous eyes were examined by selective methylmethacrylate microvascular corrosion castings following cannulation of the central retinal artery and posterior ciliary arteries in 11 normal eyes and 9 glaucomatous eyes. The resulting castings were examined with scanning electron microscopy. Microvascular changes were found in the anterior optic nerves of all the glaucomatous eyes with visual function loss. These findings include areas of capillary filling defects within the anterior optic nerve and a decreased numbers of feeding arteriolar vessels to the anterior optic nerve. In the prelaminar and laminar regions, the typical capillary patterns are lost and laminar striations are not present. Juxtapapillary choroidal and retinal avascular areas were also identified in two of the glaucomatous eyes. Selective microvascular corrosion casting is an excellent method to examine the three-dimensional microvasculature of the anterior optic nerve. Microvascular changes in the anterior optic nerve may play a role in the development of glaucomatous optic neuropathy.

Aged↗
PubMed1999

Screening of the gene encoding the alpha'-subunit of cone cGMP-PDE in patients with retinal degenerations.

PURPOSE: To screen the exons of the gene encoding the alpha'-subunit of cone cyclic guanosine monophosphate (cGMP>phosphodiesterase (PDE6C) for mutations in a group of 456 unrelated patients with various forms of inherited retinal disease, including cone dystrophy, cone-rod dystrophy, macular dystrophy, and simplex/multiplex and autosomal recessive retinitis pigmentosa. METHODS: The 22 exons of the PDE6C gene were screened for mutations either by denaturing gradient gel electrophoresis and single-strand conformation polymorphism electrophoresis (SSCP) or by SSCP alone; variants were sequenced directly. RESULTS: Although many sequence variants were found, none could be associated with disease. CONCLUSIONS: The results show that PDE6C was not the site of the amutations responsible for the types of inherited retinal degenerations analyzed in the large population of patients 'in the present study. The types of degeneration included those that predominantly affect cone-mediated function (cone and cone-rod dystrophies) or rod-mediated function (retinitis pigmentosa) or that have a predilection for disease in the macula (macular dystrophies).

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1998

Exon screening of the genes encoding the beta- and gamma-subunits of cone transducin in patients with inherited retinal disease.

PURPOSE: To screen the exons of the genes encoding the beta3-subunit (GNB3) and gammac-subunit (GNGT2) of cone transducin for mutations in a large number of unrelated patients with various forms of inherited retinal disease including cone dystrophy, cone-rod dystrophy and macular dystrophy. METHODS: Exons of the two genes were screened for mutations by denaturing gradient gel electrophoresis (DGGE) and/or single strand conformation polymorphism electrophoresis (SSCP); any variants were sequenced directly. RESULTS: Although many sequence variants were found in both genes, none could be associated with disease. Additionally, the gene structure and sequence of the coding exons of GNB3 were determined and compared with those of the dog homolog. Both human and canine GNB3 have nine coding exons and their two predicted amino acid sequences have 97% identity. CONCLUSIONS: The results indicate that GNB3 and GNGT2 are unlikely sites of mutations responsible for inherited retinal degenerations that predominantly effect cone-mediated function (cone and cone-rod dystrophies) or have a predilection for disease in the macula (macular dystrophies).

Amino Acid Sequence↗
PubMed1996

Perforin, Fas ligand, and tumor necrosis factor are the major cytotoxic molecules used by lymphokine-activated killer cells.

Lymphokine-activated killer (LAK) cells generated from perforin knockout mice possess significantly reduced cytotoxicity against a panel of tumor target cell lines, with some tumor cells being lysed exclusively by the perforin pathway. LAK cells are also capable of Fas ligand-mediated cytotoxicity. LAK cells generated from mice deficient in both perforin and Fas ligand (PKO/gld) were not cytolytic in short term cytotoxicity assays, demonstrating that perforin and Fas ligand are required for acute target cell lysis. However, PKO/gld LAK cells were cytotoxic in long term cytotoxicity assays against TNF-sensitive tumor lines, and this cytotoxicity was completely inhibited by neutralizing TNF Abs. This potent TNF cytotoxicity has not been fully appreciated previously because of the presence of dominant-acting perforin and Fas ligand in acute tumor cell lysis. TNF-based cytotoxicity by PKO/gld LAK was both soluble and membrane bound, and both forms of TNF were constitutively expressed. Thus, LAK cells are armed with at least three cytotoxic molecules: perforin, Fas ligand, and TNF.

Animals↗
PubMed1996

Screening of the PDE6B gene in patients with autosomal dominant retinitis pigmentosa.

Each of the 22 exons and 140 bp of the 5' untranslated region of the gene encoding the beta-subunit of cGMP-phosphodiesterase (PDE6B) were screened by denaturing gradient gel electrophoresis for mutations in the DNAs of 54 unrelated individuals with autosomal dominant retinitis pigmentosa. Six different sequence variants were found in seven patients. Four of the sequence variants did not segregate with disease in the families of the respective probands and/or were present in control DNAs. The remaining two sequence variants, a Leu228His missense in exon 3 and a G to A transition in the tenth base of the splice acceptor site of intron 8, were both present in the same proband. One or the other of the two sequence variants was present in each affected member of the proband's small family and neither sequence variant was present in the one unaffected member nor in 75 unrelated controls. However, no effect on splicing of mRNA was observed in expression studies of DNA constructs containing the G to A transition. Therefore, mutations in PDE6B could not be shown to be the cause of adRP in this group of patients.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1995

Mutations in the PDE6B gene in autosomal recessive retinitis pigmentosa.

We have studied 24 small families with presumed autosomal recessive inheritance of retinitis pigmentosa by a combination of haplotype analysis and exon screening. Initial analysis of the families was made with a dinucleotide repeat polymorphism adjacent to the gene for rod cGMP-phosphodiesterase (PDE6B). This was followed by denaturing gradient gel electrophoresis (DGGE) and single-strand conformation polymorphism electrophoresis (SSCPE) of the 22 exons and a portion of the 5' untranslated region of the PDE6B gene in the probands of each family in which the PDE6B locus could not be ruled out from segregating with disease. Two probands were found with compound heterozygous mutations: Gly576Asp and His620(1-bp del) mutations were present in one proband, and a Lys706X null mutation and an AG to AT splice acceptor site mutation in intron 2 were present in the other. Only the affecteds of each of the two families carried both corresponding mutations.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1994

Assay for maprotiline in human serum with improved sensitivity and selectivity.

The use of a photoreactor and fluorescence detection enables measurement of the tetracyclic antidepressant drug 3-(9,10-dihydro-9,10-ethanoanthracene-9-yl)-N-methylpropylamine (maprotiline) with a sensitivity of 100 pg/ml serum. This detection system is highly specific and enables the measurement of very low concentrations in the presence of high concentrations of other drugs that are often found in patient samples. The mean free portions of maprotiline and desmethylmaprotiline were found to be 2.2% and 1.5%, respectively.

Half-Life↗
PubMed1993

Suppression by HIV of IL-1 and IL-6 secretion in accessory cells: AC function defect partially corrected with exogenous IL-1 and IL-6.

To determine the effect of HIV infection on the accessory cell function of monocytes we measured the ability of HIV-infected monocytes to restore PHA-induced and soluble anti-CD3-induced T cell blastogenesis. These T cells were highly purified and depleted of monocytes (< 0.5%) and activated T cells. Monocytes were isolated using gelatin-fibronectin-coated flasks (< 1% T cells) and after 4 days in culture with granulocyte/macrophage-colony stimulating factor, they were infected with HIV. Accessory cell (AC) function was tested 2 and 7 days later, employing autologous cryopreserved T lymphocytes. Monocytes infected with HIV for 2 days lacked the ability to permit phytohemagglutinin (PHA) and anti-CD3-induced T cell blastogenesis. Noninfected monocytes restored the proliferative response of purified T cells. Interleukin 1 (IL-1) and interleukin 6 (IL-6) levels in culture supernatants were low when compared to cultures with noninfected AC. Preincubation of monocytes with human anti-HIV neutralizing antibodies did not restore either of the responses. AC treated with heat-inactivated HIV had normal accessory cell function. The addition of IL-1 and/or IL-6 partially restored the AC function for PHA stimulation, but not for anti-CD3 stimulation. We conclude that HIV infection of monocytes suppressed their accessory cell function in the T cell blastogenesis assay. The response was partially restored with IL-1 and/or IL-6, suggesting that HIV infection down-regulated the monocyte production of both cytokines.

Antigen-Presenting Cells↗
PubMed1991

Phosphorylation of aortic plasma membranes by protein kinase C.

Incubation of plasma membranes isolated from bovine aorta with either 0.5 mM CaCl2 or with a phorbol ester (1 microM phorbol 12,13-dibutyrate) and phosphatidylserine in an EGTA-containing buffer resulted in the phosphorylation of 10 proteins (Mr of 158, 105, 75, 62, 44, 39, 33, 22, 15 and 9 kDa), presumably due to activation of endogenous protein kinase C (PKC). After heat treatment of the aortic plasma membranes at 80 degrees C for 5 min in order to inactivate all endogenous protein kinase, phosphatase and ATPase activities, membrane phosphorylation was absolutely-dependent upon the addition of an exogenous, partially-purified PKC preparation from bovine aorta. Under these conditions, a total of 17 phosphoproteins could be detected (Mr of 158, 105, 75, 44, 39, 33, 30, 29, 27, 25, 22, 17.5, 16, 15, 11, 10 and 9 kDa). The most prominent phosphoprotein band in native membranes had a molecular weight of 75 kDa (p75); several characteristics suggest that p75 might be autophosphorylated PKC. The phosphorylation of aortic plasma membranes by exogenous PKC required phosphatidylserine and was calcium-dependent (10(-5) to 10(-7) M Ca2+); the addition of diolein resulted in little or no enhancement of phosphorylation. Replacement of phosphatidylserine with oleic acid resulted in the same number of phosphoproteins, but the extent of phosphorylation was diminished. The phosphorylation pattern was altered slightly if the aortic plasma membranes were isolated in the presence of 1 mM Ca2+ instead of EGTA buffers as in the standard procedure. Experiments were performed to determine if the p39 substrate of PKC in aortic plasma membranes was calpactin II (lipocortin I). Immunoblotting established that calpactin II was present in aortic plasma membranes, but there was no corresponding phosphoprotein on the autoradiographs.

Animals↗
PubMed1991

Calmodulin inhibits the protein kinase C-catalysed phosphorylation of an endogenous protein in A10 smooth-muscle cells.

The protein kinase C (PKC) activator phorbol 12,13-dibutyrate stimulated the phosphorylation of a 75 kDa protein (p75) in intact cultured A10 smooth-muscle cells and sonicated cell preparations; p75 was the only major substrate for endogenous PKC in sonicated A10 cells. The Ca(2+)-dependent phosphorylation of p75 in vitro was dramatically decreased in PKC-down-regulated A10 cells; however, p75 from identical sonicated cell preparations was still phosphorylated by an exogenous aortic PKC preparation. Calmodulin inhibited the phosphorylation of p75 by PKC, but not the phosphorylation of other PKC substrates (platelet P47 protein and histone). The addition of calmodulin after the phosphorylation reaction was started prevented further phosphorylation, but did not decrease the extent of phosphorylation of p75 that was reached before the addition of calmodulin. The inhibition of p75 phosphorylation was concentration-dependent, with IC50 values (concn. giving 50% inhibition) ranging from less than 0.5 to 10 micrograms of calmodulin/ml, and was Ca(2+)-dependent, requiring a free Ca2+ concentration of 10 microM or greater. These results suggest that the inhibition of the PKC-catalysed phosphorylation of p75 by calmodulin may be due to its interaction with the substrate, rather than a direct inhibitory effect on the enzyme, and that this inhibition could be regulated by intracellular Ca2+ concentration. Therefore, p75 may be a physiological link between the PKC and Ca2+/calmodulin pathways.

Animals↗