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Biomedical subjects

Y Ma

Publications and source records attributed to Y Ma.

At least 397 records · Page 22Linked to original sources
PubMed1996

More sensitive way to determine iron using an iron(II)-1,10-phenanthroline complex and capillary electrophoresis.

Iron is one of the major metal species of concern in many samples, such as in serum, foods, drinking waters, etc. In this paper, we present a more sensitive way to determine the iron concentration in water solutions by using an iron(II)-1,10-phenanthroline complexing system with high-performance capillary electrophoresis, and have applied this method to the determination of the levels of iron in serum samples. The technique uses ammonium acetate-acetic acid (50 mM NH4Ac-HAc, pH 5.0) as a running buffer, and the detection wavelength is set at 270 nm instead of 508 nm. This new approach enhances the molar absorbance of the Fe(II)-1,10-phenanthroline complex by about eight-fold compared with that obtained at 508 nm. By combining the larger light output of the deuterium (D2) lamp and the lower noise level at 270 nm, the sensitivity was improved at least twenty-fold compared to that at 508 nm. The detection limit for iron(II) is lower than 5 x 10(-9) M, which has never been reached by reported spectrophotometric methods or with the recently published HPCE method. The effects of pH, buffer concentration and operation voltages on the sensitivity and resolution are also discussed. The signal response is linear over two orders of magnitude (r2 = 0.995) and the iron recovery for samples reached 99-101%. The technique described here is much more sensitive, fast and simple and is suitable for determining trace amounts of iron in biological, food, water and other samples.

Buffers↗
PubMed1996

Hedgehog patterning activity: role of a lipophilic modification mediated by the carboxy-terminal autoprocessing domain.

Autocatalytic processing mediated by the carboxyterminal domain of the hedgehog (hh) protein precursor (Hh) generates an amino-terminal product that accounts for all known signaling activity. The role of autoprocessing biogenesis of the hh signal has been unclear, since a truncated unprocessed protein lacking all carboxy-terminal domain sequences retains signaling activity. Here, we present evidence that the autoprocessing reaction proceeds via an internal thioester intermediate and results in a covalent modification that increases the hydrophobic character of the signaling domain and influences its spatial and subcellular distribution. We demonstrate that truncated unprocessed amino-terminal protein causes embryonic mispatterning, even when expression is localized to cells that normally express Hh, thus suggesting a role for autoprocessing in spatial regulation of hh signaling. This type of processing also appears to operate in the biogenesis of other novel secreted proteins.

Animals↗
PubMed1996

Adenovirus-mediated gene transfer into rat cardiac allografts. Comparison of direct injection and perfusion.

With the ultimate goal of modulating the host immune response in organ transplantation, gene therapy studies have demonstrated that direct plasmid DNA injection into transplanted myocardium can result in detectable levels of transgene expression. However, the restricted distribution and low level of transgene expression evident in these studies have limited its application. Recently, replication-defective adenovirus vectors have been shown to be an efficient gene-transfer vehicle in vivo whose infection does not require target-cell proliferation. In the present study, adenovirus vectors encoding reporter genes were delivered into transplanted hearts by either direct injection into the myocardium or perfusion via aorta of the donor hearts. The efficacy and stability of the transgene expression by perfusion and by direct injection were examined and compared. Using the adenovirus vector encoding the firefly luciferase gene, we found that a higher level of transgene expression was achieved by direct injection, but that more evenly distributed transgene expression was observed in hearts perfused with viral vector. These results were further confirmed by 5-bromo-4-chloro-3-indolyl-beta-d-galactoside histochemical staining of another adenoviral vector encoding beta-galactosidase. The transgene expression was not stable and decreased within 1 month with either delivery method. Nevertheless, these results indicate that adenovirus-mediated gene transfer can result in short-term expression of the gene throughout the heart and may be useful as a gene vector in organ transplantation.

Adenoviridae↗
PubMed1996

Affinity countercurrent chromatography using a ligand in the stationary phase.

In countercurrent chromatography (CCC), an addition of a ligand to the liquid stationary phase remarkably improved both retention time and peak resolution of the analytes: various amino acid derivatives were separated by N-dodecanoyl-L-proline-3,5-dimethylanilide, while polar catecholamines and dipeptides were separated by bis-(2-ethylhexyl)phosphoric acid. By selecting an appropriate ligand and dissolving it in the liquid stationary phase, the present CCC technique can perform a variety of separations comparable to chiral chromatography, ion chromatography, and affinity chromatography. Leakage of the ligand from the column can be entirely eliminated by introducing a small volume of a ligand-free stationary phase at the end of the column as an absorbent. The method further facilitates application of pH-zone-refining CCC and can increase the sample loading capacity over 10 times for a given column.

Amino Acids↗
PubMed1996

Selective upregulation of T alpha 1 alpha-tubulin and neuropeptide Y mRNAs after intermittent excitatory stimulation in adult rat hippocampus in vivo.

Adult central neurons exhibit significant structural and molecular changes in epilepsy. We have examined changes in two markers of morphological and physiological plasticity, T alpha 1 alpha-tubulin (T alpha 1) and neuropeptide Y (NPY) mRNAs, in response to intermittent (20 Hz, 10 seconds, 1 minute-1) stimulation of the rat perforant path in vivo. Stimulus trains elicited brief (0.5-3 seconds) afterdischarges in the ipsilateral dentate gyrus (DG). Four hours of stimulation caused no significant loss of inhibition in the DG 40-48 hours after stimulation ceased. However, it did lead to an increase in NPY mRNA in neurons of the ipsilateral and, to a lesser extent, contralateral DGs and Ammon's Horn. Many of these were presumably interneurons that normally express NPY. However, dentate granule cells (DGCs), which do not normally express this peptide, also expressed robust levels of NPY mRNA bilaterally. NPY mRNA levels peaked at 4-24 hours and returned to baseline by 48 hours poststimulation. Although 24 hours of stimulation induced a similar increase in interneurons, DGCs showed no detectable NPY mRNA. Afterdischarges were necessary to elevate NPY mRNA expression. Four hours of stimulation elevated T alpha 1 mRNA expression in both ipsilateral and, to a lesser extent, contralateral DGCs; this elevation peaked at 24 hours poststimulation and declined to baseline by 72 hours. Stimulation for 24 hours caused broader changes in T alpha 1 mRNA expression, with increases in DGCs and in CA3 pyramidal cells bilaterally. Acute denervation of the DG did not affect T alpha 1 mRNA level in the hippocampal formation. Elevated synaptic input resulting in afterdischarges, but not necessarily in excitability changes in the DG, led to alterations in the expression of molecular markers of plasticity. These changes may reflect adaptive responses to physiological activation.

Animals↗
PubMed1996

A mutation in the lipoprotein lipase gene is the molecular basis of chylomicronemia in a colony of domestic cats.

Members of a domestic cat colony with chylomicronemia share many phenotypic features with human lipoprotein lipase (LPL) deficiency. Biochemical analysis reveals that these cats do have defective LPL catalytic activity and have a clinical phenotype very similar to human LPL deficiency. To determine the molecular basis underlying this biochemical phenotype, we have cloned the normal and affected cat LPL cDNAs and shown that the affected cat has a nucleotide change resulting in a substitution of arginine for glycine at residue 412 in exon 8. In vitro mutagenesis and expression studies, in addition to segregation analysis, have shown that this DNA change is the cause of LPL deficiency in this cat colony. Reduced body mass, growth rates, and increased stillbirth rates are observed in cats homozygous for this mutation. These findings show that this LPL deficient cat can serve as an animal model of human LPL deficiency and will be useful for in vivo investigation of the relationship between triglyceride rich lipoproteins and atherogenic risk and for the assessment of new approaches for treatment of LPL deficiency, including gene therapy.

Amino Acid Sequence↗
PubMed1996

In vitro import of pre-ferredoxin-NADP+-oxidoreductase from Cyanophora paradoxa into cyanelles and into pea chloroplasts.

Using a novel cyanelle isolation procedure we showed that pre-ferredoxin-NADP+-oxidoreductase (pre-FNR) from C. paradoxa is translocated in vitro across the peptidoglycan-containing cyanelle envelope. Efficient import was also observed in a heterologous system with pea chloroplasts as the recipient organelles. These results support the conclusion derived from comparative analysis of plastid genome organization, that all plastids originate from a common semi-autonomous endosymbiotic ancestor.

Cell Fractionation↗
PubMed1996

pH-Zone refining counter-current chromatography of polar catecholamines using di-(2-ethylhexyl)phosphoric acid as a ligand.

The use of di-(2-ethylhexyl)phosphoric acid (DEHPA) as a ligand in the stationary phase effectively increased the partition coefficient of polar catecholamines. pH-Zone refining counter-current chromatography of six components, i.e. five catecholamines and one amino acid (DOPA), was successfully performed using a two-phase solvent system composed of methyl tert.-butyl ether and water by adding DEHPA (20%) and ammonium acetate (200 mM) to the organic phase and HCl (50 mM) to the aqueous mobile phase. DOPA was eluted first as a normal peak followed by the five catecholamines which formed a succession of highly concentrated rectangular peaks associated with sharp impurity peaks at their borders (UV tracing at 280 nm). Both pH and standard partition coefficient of collected fractions indicated minimum overlap between the main peaks. Each component was identified by NMR analysis.

Catecholamines↗
PubMed1996

Isolation of anti-HIV-1 lignans from Larrea tridentata by counter-current chromatography.

Several lignans, mostly new, were isolated from Larrea tridentata by assay-guided counter-current chromatography (CCC). Using the secreted alkaline phosphatase bioassay of HIV Tat transactivation and the two-phase hexane-ethyl acetate-methanol-water solvent system, two major components (Gr and Lo) were identified as anti-HIV active principles. The chemical structures of the constituents of Gr (G1-G4) and Lo (L1-L4) were determined by GC-MS and NMR. After optimization of isolation conditions, a large-scale isolation with the chloroform-methanol-water system yielded five constituents (FB1-FB5). The most predominant anti-HIV compound FB2 (denoted Malachi 4:5-6 or mal.4), which occurs in 0.23% yield, was separated from its FB1 isomer (0.13% yield). Compound FB4 and two tricyclic lignans (FB3 and FB5) were also isolated in a substantial amount for further testing of their anti-HIV activities. These compounds may represent a new class of anti-HIV agents with important clinical relevance.

Antiviral Agents↗
PubMed1996

Distinct temporal patterns of defensin mRNA regulation during drug-induced differentiation of human myeloid leukemia cells.

Defensins are microbicidal peptides and the principal constituents of neutrophil primary granules. They are presumed to play a prominent role in innate host defenses. We examined defensin mRNA levels during drug-induced differentiation of the promyelocytic leukemia cell line, HL-60. Transcription was restricted to promyelocyte, myelocyte, and very early metamyelocyte stages of the granulocytic pathway. Complete downregulation occurred during late granulocytic maturation or early during phorbol ester-promoted differentiation along the monocyte/macrophage lineage. Retinoic acid (RA) was the strongest inducer of defensin mRNA accumulation, even at doses too low to effect morphologic changes; the initial (first 48 hours), gradual increase resulted from transcriptional activation and was enhanced by granulocyte colony-stimulating factor. In contrast, addition of hybrid polar compounds led to a transient, drug-specific downregulation within the same time period, apparently by means of selectively induced, biphasic degradation of transcripts. Subsequent increase in transcript levels was faster and more pronounced with hexamethylene bisacetamide, relative to dimethyl sulfoxide (DMSO). DMSO-promoted effects were strikingly different in serum-free medium or in the presence of the tyrosine kinase inhibitor, genistein. Under these conditions, and although differentiation was unaffected, early defensin mRNA downregulation was final. The effect did not occur with RA and expression of other myeloid-specific genes was also unchanged. Addition of selected cytokines caused a similar "dip," only at earlier times and uncoupled from differentiation. Tumor necrosis factor-alpha markedly induced defensin levels after 2 days in previously untreated HL-60 cells, but inhibited expression in RA-differentiated cells. These results begin to detail a complex regulation of defensin mRNA synthesis with both spatial and temporal control elements, and a unique modulation by chemical agents, cytokines, and serum-factors.

Acetamides↗
PubMed1996

Control of the production of soluble interleukin-4 receptors: implications in immunoregulation.

Soluble cytokine receptors (sCR) are generated in vivo through proteolytic cleavage of the membrane-bound receptors or by direct translation of mRNAs specifically encoding the soluble forms. Despite their widespread presence in biological fluids, the physiological role of endogenous sCR as immunoregulatory molecules is not yet well understood. In vivo, exogenous soluble interleukin-4 receptors (sIL-4R) have been shown to have both agonistic and antagonistic effects on IL-4 responses, depending on the relative concentration ratios of sIL-4R to IL-4. In an effort to elucidate the potential role of endogenous sIL-4R in the regulation of IL-4 responses, the mechanisms controlling the production of sIL-4R have been investigated. Although many cell types are able to constitutively produce low levels, production of sIL-4R is significantly up-regulated in vitro by T cell activation and IL-4. The ability of splenic cells to produce sIL-4R and the serum levels of sIL-4R have consistently been found to be increased during immune responses characterized by T cell activation and IL-4 secretion (Th2 responses). In agreement, clones of Th2, but not Th1, cells were found to significantly up-regulate sIL-4R production following antigenic stimulation. However, the production of sIL-4R by Th2 cells appears to be independent from that of IL-4 and can also be induced by cell contact and/or IL-1-dependent pathways. Taken together, these observations suggest that the production of sIL-4R in vivo is closely associated with the secretion of IL-4, and are consistent with the notion that endogenous sIL-4R are involved in the regulation of IL-4 activity during immune responses.

Animals↗
PubMed1996

Compound heterozygosity for a known and a novel defect in the lipoprotein lipase gene (Asp250-->Asn; Ser251-->Cys) resulting in lipoprotein lipase (LPL) deficiency.

Two missense mutations in exon 6 of the LPL gene were identified on separate alleles in a Dutch patient with lipoprotein lipase (LPL) deficiency. The first mutation is a G1003-->A transition resulting in a D250N mutation, which has been shown previously to result in a catalytically defective protein in patients of French-Canadian ancestry. The second mutation, a C to G transition at nucleotide 1007, predicts a S251C residue change in the highly conserved region of LPL surrounding the loop structure the covers the catalytic triad. This mutation constitutes a novel defect among LPL gene mutations reported so far. Site-directed mutagenesis experiments provide in-vitro evidence for the complete loss of LPL activity resulting from this latter missense mutation. The G1003-->A nucleotide substitution underlying the Asp250 mutation deletes a TaqI endonuclease recognition site and the C1007-->G change that leads to the S251C alteration abolishes a HinfI recognition site. This will facilitate rapid screening for these mutations in LPL-deficient patients.

Adolescent↗
PubMed1996

Ginseng extract scavenges hydroxyl radical and protects unsaturated fatty acids from decomposition caused by iron-mediated lipid peroxidation.

This study was conducted to investigate whether or not the antioxidation effect of ginseng extract directly inhibits decomposition of unsaturated fatty acid caused by iron and hydrogen peroxide-induced lipid peroxidation, and whether this effect involves a hydroxyl radical-scavenging mechanism. Thiobarbituric acid-reactive substances (TBARS), gas chromatography, and electron spin resonance (ESR) spectrometer were used to measure lipid peroxidation, unsaturated fatty acid, and hydroxyl radical. The results showed TBARS formed and the loss of arachidonic acid during lipid peroxidation, and that hydroxyl radical formed by the Fenton reaction were completely inhibited by ginseng extract. This antioxidant effect of ginseng may be responsible for its wide pharmacological actions in clinical practice by a free radical reaction-inhibition mechanism.

Arachidonic Acid↗
PubMed1996

Combined flurbiprofen and cyclosporin-A does not attenuate bone loss and exaggerates renal impairment.

Cyclosporine (CsA) is a potent immunosuppressant that has revolutionized the success of organ transplantation. Flurbiprofen (FB), a propionic acid derivative NSAID, has been demonstrated in vivo to reduce osteoclast numbers in normal rats. The aim of this experiment was to determine whether addition of FB to CsA-treated rats could prevent the bone changes associated with CsA therapy. Forty-eight 10-12-week-old male Sprague-Dawley rats were randomized to receive, daily for 28 days: (1) CsA vehicle p.o. plus FB vehicle sc; (2) CsA (15 mg/kg) p.o. plus FB vehicle sc, (3) CsA vehicle p.o. plus FB (1.5 mg/kg) sc; and (4) CsA (15 mg/kg) p.o. plus FB (1.5 mg/kg) sc. Rats were weighed and venous blood sampled at baseline, 14 days, and 28 days for determination of glucose, Ca+2, BUN, creatinine, PTH, osteocalcin, and 1,25(OH)2 vitamin D. Tibiae were removed following killing, after double labeling for histomorphometry. Body mass was significantly lower than control in all rats receiving CsA on days 14 and 28 while blood glucose was only elevated in the CsA alone group. Day 28 BUN and creatinine were significantly elevated in the CsA group and the combination of CsA and FB revealed an exacerbation of this trend. Vitamin D and osteocalcin were consistently increased in the CsA and CsA/FB groups. Bone histomorphometry showed evidence of trabecular osteopenia in CsA and CsA/FB groups. CsA alone resulted in elevated bone turnover. FB was unable to prevent the trabecular bone loss induced by CsA therapy. This experiment indicates no role for FB as a therapeutic option in CsA-induced bone disease at the given doses and duration of treatment by virtue of its lack of bone sparing ability and adverse renal effects when the two drugs are administered concurrently.

Administration, Oral↗