Biomedical subjects

F Mao

Publications and source records attributed to F Mao.

16 recordsLinked to original sources

PubMed1997

Developmental expression and regulation of alpha 1 adrenergic receptors in cultured cortical neurons.

The expression and distribution of alpha 1 receptors in cultured neurons derived from rat visual cortex were studied with confocal microscopy using the fluorescently labeled alpha 1 adrenergic receptor selective antagonist BODIPY FL prazosin. The receptors were found to be clustered on neuronal somata and on proximal dendrites. We found that expression of the alpha 1 receptor is regulated both by neuronal excitability and by the usage of the receptor itself. The specificity of receptor regulation to the blockade of a particular receptor class was also studied. We approached this by comparing the effects of treatment with the alpha 1 adrenoreceptor antagonist prazosin and M1 muscarinic receptor antagonist pirenzepine on the expression of alpha 1 and M1 receptors in cultured cortical neurons. The results showed that blockade of muscarinic receptors with pirenzepine up-regulated muscarinic receptor expression selectively without changing alpha 1 receptor expression. Conversely blockade of alpha 1 receptors up-regulated alpha 1 expression but not muscarinic receptor expression. This implies that the expression levels of M1 and alpha 1 receptors are both regulated through specific signal transduction pathways. The interactions between neuronal activity and receptor activation (or blockade) on receptor expression were studied as well. In these experiments we compared the effects of high K+, tetrodotoxin (TTX), prazosin, and noradrenaline on the expression of alpha 1 and M1 receptors in cultured neurons. The results show that high K+ exposure increased both alpha 1 and M1 receptor expression regardless of the presence of receptor agonists or antagonists. On the other hand, TTX exposure reduced both alpha 1 and M1 receptor expression regardless of the presence of the receptor agonists or antagonists. This implies that regulation of receptor number is predominantly regulated by neuronal activity rather than by receptor occupancy.

Animals↗

PubMed1997

Cholesterol deposition in macrophages: foam cell formation mediated by cholesterol-enriched oxidized low density lipoprotein.

Oxidized low density lipoprotein (LDL) is thought to mediate the transformation of macrophages to cholesterol-rich foam cells. Yet convincing evidence for this process is lacking in vitro. We suggest that oxidized LDL-mediated foam cell formation is not seen in vitro because the cholesteryl ester content of LDL particles (oxidized in the presence of transition metals) is dramatically reduced. Thus, if oxidized LDL could be cholesterol-enriched prior to its addition to macrophages, this lipoprotein would be made more capable of inducing the cellular deposition of cholesteryl esters. When we enriched cupric sulfate-oxidized LDL with cholesterol by incubation of this lipoprotein with unesterified cholesterol/phosphatidylcholine liposomes and added it to mouse peritoneal macrophage cultures, we found that: a) the enrichment of oxidized LDL with cholesterol did not alter the extent of oxidized LDL degradation; b) the cells accumulated massive amounts of cholesteryl ester (148 microg/mg cell protein) and unesterified cholesterol (260 microg/mg cell protein) after 24 h of incubation; and c) Sephacryl S-1000 chromatography of the cholesterol-enriched oxidized LDL verified the formation of large oxidized LDL-unesterified cholesterol/phosphatidylcholine complexes. These results demonstrate that oxidized LDL, when cholesterol-enriched, can mediate the formation of macrophage foam cells in culture

Animals↗

PubMed1996

Selective induction of apoptosis in Hep 3B cells by topoisomerase I inhibitors: evidence for a protease-dependent pathway that does not activate cysteine protease P32.

Progress in the treatment of hepatocellular carcinoma (HCC), a common tumor worldwide, has been disappointing. Inhibitors of topoisomerases are being widely studied as potential inducers of tumor cell apoptosis. Our aims were to determine whether topoisomerase-directed drugs would induce apoptosis in a human HCC cell line (Hep 3B) and, if so, to investigate the mechanism. The topoisomerase I poison camptothecin (CPT) induced apoptosis of Hep 3B cells in a time- and concentration-dependent manner. In contrast, the topoisomerase II poison etoposide failed to induce apoptosis despite the apparent stabilization of topoisomerase II-DNA complexes. Unexpectedly, CPT-induced apoptosis in this cell type occurred without any detectable cleavage of poly(ADP-ribose) polymerase or lamin B, polypeptides that are commonly cleaved in other cell types undergoing apoptosis. Likewise, Hep 3B cell apoptosis occurred without a detectable increase in interleukin-1beta-converting enzyme (ICE)-like or cysteine protease P32 (CPP32)-like protease activity. In contrast, trypsin-like protease activity (cleavage of Boc-Val-Leu-Lys-chloromethylaminocoumarin in situ) increased threefold in cells treated with CPT but not etoposide. Tosyl-lysyl chloromethyl ketone inhibited the trypsin-like protease activity and diminished CPT-induced apoptosis. These data demonstrate that (a) apoptosis is induced in Hep 3B cells after stabilization of topoisomerase I-DNA complexes but not after stabilization of topoisomerase II-DNA complexes as measured by alkaline filter elution; (b) Hep 3B cell apoptosis occurs without activation of ICE-like and CPP32-like protease activity; and (c) a trypsin-like protease activity appears to contribute to apoptosis in this cell type.

Apoptosis↗

PubMed1996

Iron-ascorbate-phospholipid mediated modification of low density lipoprotein.

LDL can be oxidized by a variety of agents to form a modified lipoprotein which is capable of being avidly metabolized by macrophages. While previous in vitro studies have focused exclusively on the oxidation of LDL, other lipids found in the atheroma are also subject to oxidation and its lipoperoxide byproducts may contribute to the process of LDL modification. To examine the relationship between the oxidation of phospholipids and the subsequent modification of LDL, we incubated 250 microM phosphatidylcholine with 10 microM ferrous sulfate and 50 microM ascorbic acid in 10 mM Tris (pH 7.0). After 18 h at 37 degrees C, significant amounts of thiobarbituric acid reactive substances (TBARS) were formed. The inclusion of LDL (100 micrograms protein/ml) elevated the TBARS and increased the electrophoretic mobility of the lipoprotein. LDL treated with iron and ascorbate in the absence of phosphatidylcholine did not result in the modification of this lipoprotein. LDL that was incubated with phosphatidylcholine, iron and ascorbate was found to be metabolized by macrophages to a far greater extent than native LDL or LDL treated with phosphatidylcholine alone. Probucol (10 microM) inhibited the LDL modification process. These results demonstrate that while iron and ascorbate cannot oxidize LDL directly, the addition of phosphatidylcholine to these initiators of lipid peroxidation can mediate and lead to the modification of LDL.

Animals↗

PubMed1996

Characterization of the insulin-regulated membrane aminopeptidase in 3T3-L1 adipocytes.

A novel membrane aminopeptidase has been identified as a major protein in vesicles from rat adipocytes containing the glucose transporter isotype Glut4. In this study we have characterized this aminopeptidase, referred to as vp165, in 3T3-L1 adipocytes. The subcellular distributions of vp165 and Glut4 were determined by immunoisolation of vesicles with antibodies against both proteins, by immunofluorescence, and by subcellular fractionation and immunoblotting. Relative amounts of vp165 at the cell surface in basal and insulin-treated cells were assayed by cell surface biotinylation. These experiments showed that vp165 and Glut4 were entirely colocalized and that vp165 increased markedly at the cell surface in response to insulin, in a way similar to Glut4. When intact cells were assayed with a novel, membrane-impermeant fluorogenic substrate for vp165, we found that insulin stimulated aminopeptidase activity at the cell surface. This observation provides direct evidence for the functional consequence of vp165 translocation.

3T3 Cells↗

PubMed1996

Imaging exocytosis and endocytosis.

From the secretion of neurotransmitters via synaptic vesicles to the expulsion of cellular waste via contractile vacuoles, exocytosis and its sequel, endocytosis, are being explored with a variety of new optical tools. Fluorescent markers, especially styryl dyes such as FM1-43 (which reversibly labels endosomal membranes), have been used to follow exo- and endocytic events in many cell types. Even though the development of new dyes is still largely empirical, some theoretical principles have emerged to guide future dye chemistry. Moreover, advances in optical imaging technology that augment conventional fluorescence microscopy are appearing. For example, interference reflection microscopy (which requires no flurophore) and total internal reflection microscopy have recently been used to observe single exocytic events at the contact point between a glass coverslip and the plasma membrane.

Animals↗

PubMed1995

Association of negatively-charged phospholipids with low-density lipoprotein (LDL) increases its uptake and the deposition of cholesteryl esters by macrophages.

LDL, the major carrier of cholesterol in blood, is poorly metabolized by macrophages. In contrast, macrophages can recognize and endocytose anionic phospholipids such as phosphatidylserine, phosphatidylglycerol and cardiolipin. Since macrophages can take up large amounts of these phospholipids, experiments were performed to ascertain whether pre-incubation of native LDL with negatively-charged phospholipids would enhance the metabolism of LDL by macrophages. When 125I-LDL was incubated with cardiolipin liposomes for 18 h at 37 degrees C before addition to macrophages, an approx. 40-fold increase of LDL metabolism by these cells was observed. Similar results were found when LDL was pre-incubated with phosphatidylserine or phosphatidylglycerol; however, pre-incubation of LDL with phosphatidylcholine liposomes did not lead to an increase of LDL metabolism. The macrophage uptake of LDL pre-incubated with cardiolipin was reduced to approx. 40% of control values in the presence of dextran sulfate and fucoidin, inhibitors of anionic phospholipid uptake. Cytochalasin D, an inhibitor of phagocytosis, reduced the lysosomal degradation of LDL pre-incubated with cardiolipin to approx. 10% of control values. When the LDL-cardiolipin mixture was chromatographed on agarose gel, two peaks containing LDL were observed in the elution profile: the first peak appeared at the void volume and the second peak was detected just ahead of native LDL. The LDL in both peaks was much more extensively metabolized by macrophages than was native LDL; the LDL in the first peak was metabolized at a rate that was 8 times the second peak. The results demonstrate that negatively-charged phospholipids can form a complex with LDL which facilitates its phagocytosis by macrophages.

Cardiolipins↗

PubMed1995

Synthesis of a photoaffinity analog of 3'-azidothymidine, 5-azido-3'-azido-2',3'-dideoxyuridine. Interactions with herpesvirus thymidine kinase and cellular enzymes.

Long term administration of 3'-azidothymidine (AZT) for the treatment of AIDS has led to detrimental clinical side effects in some patients, the biochemical causes of which are still being delineated. Base-substituted, azido-nucleotide photoaffinity analogs have routinely proven to be effective tools for identifying and characterizing nucleotide-utilizing enzymes. Therefore, we have synthesized 5-azido-3'-azido-2',3'-dideoxyuridine, which is a potential photoaffinity analog of two human immunodeficiency virus drugs, AZT and 3'azido-2',3'-dideoxyuridine. A partially purified herpes simplex virus type 1 thymidine kinase and [gamma-32P]ATP were used to make an AZT monophosphate analog, [32P]5-azido-3'-azido-2',3'-dideoxyuridine monophosphate. The photoaffinity properties of this analog were initially tested with herpes simplex virus type 1 thymidine kinase. Photoaffinity labeling of this enzyme was saturable (half-maximal, 30 microM) and could be specifically inhibited by AZT, AZT monophosphate, thymidine, and thymidine monophosphate. Photolabeling of rat liver microsomal membranes was also done, and several membrane proteins that interact with AZT monophosphate were identified. The antiviral and cytotoxic activities of 5-azido-3'-azido-2',3'-dideoxyuridine were determined using human immunodeficiency virus, type 1 strain IIIB and an AZT drug-resistant strain in human T lymphocyte H9 cells.

Affinity Labels↗

PubMed1995

Purification and photoaffinity labeling of herpes simplex virus type-1 thymidine kinase.

The molecular basis for the treatment of human herpesviruses with nucleoside drugs is the phosphorylation of these drugs by the viral-encoded thymidine kinases. In order to better understand the structural and enzymatic mechanisms by which herpesviral thymidine kinases recognize their substrates, photoaffinity labeling with [alpha-32P]5-azido-2'-deoxyuridine-5'-monophosphate and [ gamma-32P]8-azidoadenosine-5'-triphosphate was used to characterize the thymidine, thymidylate, and ATP active sites of the herpes simplex virus-1 (HSV-1) thymidine kinase. For this study, HSV-1 thymidine kinase and a site-specific mutant enzyme (C336Y, known to confer acyclovir resistance) were expressed in bacteria and purified by a rapid, two-step protocol. The specificity of photoaffinity labeling of these HSV-1 thymidine kinases was demonstrated by the ability of site-directed substrates such as thymidine, thymidylate, acyclovir, 5-bromovinyl-2'-deoxyuridine, and ATP to inhibit photoinsertion. Differences in inhibition patterns of photoaffinity labeling correlated with kinetic differences between the wild-type and C336Y HSV-1 thymidine kinases. Cumulative results suggest that the acyclovir-resistant cysteine 336 mutation primarily affects the ATP binding site; yet it also leads to alteration in the binding affinity of nucleoside drugs in the thymidine site. In this study, azidonucleotide photoaffinity analogs are shown to be effective tools for studying the active-site environment of HSV-1 thymidine kinase and related site-specific mutants.

Adenosine Triphosphate↗

PubMed1994

Optical measurements of activity-dependent membrane recycling in motor nerve terminals of mammalian skeletal muscle.

Motor nerve terminals in a variety of rat and mouse skeletal muscles were stained in an activity-dependent fashion using the styryl dyes FM1-43 or FM2-10. Low-light video microscopy and digital image processing techniques were used to evaluate destaining of the preparations during application of depolarizing stimuli. Best results were obtained with the mouse triangularis sterni muscle. Quantitative analysis of the destaining of dye-loaded terminals supports the suggestion that FM1-43 stains a recycling membrane compartment, most probably synaptic vesicles. However, the pattern of staining and destaining were not the same as those reported previously for frog neuromuscular junctions. The pattern of nerve terminal staining was less punctate and the rate and amount of activity-dependent destaining were less than in frog muscle. Part of the explanation may be a more acute susceptibility of mammalian terminals to phototoxicity.

Animals↗

PubMed1994

Activity-dependent expression and distribution of M1 muscarinic ACh receptors in visual cortex neuronal cultures.

The fluorescently labeled muscarinic M1 receptor-selective antagonist BODIPY FL pirenzepine has been employed to study the activity-dependent distribution and expression of muscarinic M1 ACh receptors (M1AChRs) in cultured neurons derived from rat visual cortex. Displacement experiments showed that like pirenzepine, binding of BODIPY FL pirenzepine was specific to M1 receptors and its K(i) was similar to that of unlabeled pirenzepine. Using confocal laser scanning microscopy, M1 receptors were predominantly localized to cell bodies early in development in the culture environment. After 2 weeks in culture, the receptors showed labeling not only in cell bodies but also in neuritic processes, especially on the initial segments of the processes. Chronic membrane depolarization with 40 mM potassium chloride caused a dramatic increase in M1 receptor expression on these neurons. Conversely, blockade of neuronal activity with 0.1 microM TTX decreased expression of the receptors. Receptor expression increased after cells were treated chronically with 50 nM pirenzepine, whereas it decreased after exposure to 10 microM carbachol. The results demonstrate for the first time the exact location of muscarinic receptors in living cultured neurons and also the activity-dependent expression of M1 receptors on these neurons. Both chronic membrane depolarization and antagonist application upregulate receptor expression, whereas blocking bioelectrical activity or chronic agonist application downregulates expression.

Animals↗

PubMed1993

An interactive program for pharmacokinetic modeling.

A computer program, PharmK, was developed for pharmacokinetic modeling of experimental data. The program was written in C computer language based on the high-level user-interface Macintosh operating system. The intention was to provide a user-friendly tool for users of Macintosh computers. An interactive algorithm based on the exponential stripping method is used for the initial parameter estimation. Nonlinear pharmacokinetic model fitting is based on the maximum likelihood estimation method and is performed by the Levenberg-Marquardt method based on chi 2 criterion. Several methods are available to aid the evaluation of the fitting results. Pharmacokinetic data sets have been examined with the PharmK program, and the results are comparable with those obtained with other programs that are currently available for IBM PC-compatible and other types of computers.

Administration, Oral↗

PubMed1992

[Studies on polypeptides. IX. Synthesis and antigen specificity of pre-S region fragments of hepatitis B surface antigen].

Seven peptide fragments of HBsAg Pre-S region have been synthesized by liquid phase and solid phase methods. All peptide fragments were linked to protein carrier by carbodiimide and glutaraldehyde methods. The antigen specificity of the peptide--protein conjugates was assayed by anti-HBsAg polyclonal antibodies and anti--"a" monoclonal antibody. P1 and P5 were found to have higher antigen specificity.

Amino Acid Sequence↗

PubMed1992

Activity-dependent fluorescent staining and destaining of living vertebrate motor nerve terminals.

Living motor nerve terminals from several species can be stained in an activity-dependent fashion by certain styryl dyes, such as RH414, RH795, and a new dye, FM1-43, which can be imaged independently of the others. The dyes evidently become trapped within recycled synaptic vesicles. In frog cutaneus pectoris muscle, bright fluorescent spots spaced regularly along the length of the nerve terminals appear after stimulation in the presence of the dye. The spots align well with postsynaptic ACh receptors and are persistent for many hours, unless further stimulation is given, in which case the spots disappear. Destaining, like staining, requires transmitter release and proceeds gradually over several minutes at high stimulus frequencies (e.g., 30 Hz), and fluorescent spots in the same terminal disappear at about the same rate. We suggest that each spot is a cluster of hundred of synaptic vesicles and that the mechanism of staining involves the ability of the dyes to partition reversibly into the outer leaflet of surface membranes, without being able to penetrate the entire membrane thickness. Then, during endocytosis following transmitter release, dye molecules become trapped in recycled synaptic vesicle membranes. The dyes therefore make it possible optically to study vesicle exocytosis and recycling in living nerve terminals in real time, and should be useful for marking terminals in a variety of preparations according to their level of activity.

Animals↗

PubMed1991

[Synthesis of new test reagents for the estrogen receptor in human breast cancer tissues].

According to the character of structure and activity of the test reagents I-IV, two new reagents V and VI for detecting estrogen receptor of human mammary cancer cells were synthesized. This simplifies the route of synthesis and increases activity. Key intermediates VIII and IX were confirmed by IR, MS, UV and elemental analysis. The quantification of the final products V and VI were determined by UV. The result of preliminary clinico-pathological test shows compound V to be effective on estrogen receptor.

Breast Neoplasms↗

PubMed1991

[Synthesis of receptor reagent--estradiol-polyethyleneglycol-fluorescein isothiocyanate].

Estradiol conjugate has been used as a tracer to detect estrogen receptor of human mammary cancer cells. In order to look for more stable carrier of fluorescent estradiol conjugate, we substituted polyethyleneglycol (PEG) for bovine serum albumin (BSA) and synthsized 17 beta-estradiol-6-carboxymethyl-oxime-PEG-fluorescein isothiocyanate. The structure of the title compound was confirmed by IR and UV. In this paper, we also describe another procedure to convert the terminal hydroxyl groups of PEG to the more reactive primary amino PEG by tosylation, azide group substitution and catalytic hydrogenation. The ratio of conversion is about 87%.

Estradiol↗