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Biomedical subjects

L Tsai

Publications and source records attributed to L Tsai.

At least 19 recordsLinked to original sources
PubMed2000

Modifications of proteins by polyunsaturated fatty acid peroxidation products.

The ability of unsaturated fatty acid methyl esters to modify amino acid residues in bovine serum albumin (BSA), glutamine synthetase, and insulin in the presence of a metal-catalyzed oxidation system [ascorbate/Fe(III)/O(2)] depends on the degree of unsaturation of the fatty acid. The fatty acid-dependent generation of carbonyl groups and loss of lysine residues increased in the order methyl linoleate < methyl linolenate < methyl arachidonate. The amounts of alkyl hydroperoxides, malondialdehyde, and a number of other aldehydes that accumulated when polyunsaturated fatty acids were oxidized in the presence of BSA were significantly lower than that observed in the absence of BSA. Direct treatment of proteins with various lipid hydroperoxides led to a slight increase in the formation of protein carbonyl derivatives, whereas treatment with the hydroperoxides together with Fe(II) led to a substantial increase in the formation of protein carbonyls. These results are consistent with the proposition that metal-catalyzed oxidation of polyunsaturated fatty acids can contribute to the generation of protein carbonyls by direct interaction of lipid oxidation products (alpha,beta-unsaturated aldehydes) with lysine residues (Michael addition reactions) and also by interactions with alkoxyl radicals obtained by Fe(II) cleavage of lipid hydroperoxides that are formed. In addition, saturated aldehydes derived from the polyunsaturated fatty acids likely react with lysine residues to form Schiff base adducts.

Animals↗
PubMed2000

p39 activates cdk5 in neurons, and is associated with the actin cytoskeleton.

Cyclin-dependent kinase 5 (cdk5) is a small serine/threonine kinase that displays close sequence homology to the mitotically active cyclin-dependent kinases. Cdk5 has been shown to play an essential role in the development of the nervous system, including neuronal migration and neurite outgrowth. Cdk5 activation requires the presence of a regulatory activator such as p35. cdk5 -/- mice have much more extensive defects in the development of the nervous system than p35 -/- mice, leading to the speculation that other regulatory activators of cdk5 exist. Indeed, p39 is a p35 related protein isolated by sequence homology to p35. We show here that p39 associates with cdk5 in brain lysates, and that this complex is active in phosphorylation of histone H1. By extensive characterization of p39 subcellular localization in different cell types, we demonstrate the presence of p39 in lamellipodial and fillopodial structures of cells and in growth cones of neurons. We show that p39 colocalizes with actin, and cofractionates with the detergent insoluble cytoskeleton from brain. Further, p39 coimmunoprecipitates with actin in brain lysates. Finally, disruption of the actin cytoskeleton alters p39 subcellular localization as well as kinase activity of the p39/cdk5 complex. Therefore, our results reveal the existence of the p39/cdk5 complex in vivo and suggest that it might play a role in regulating actin cytoskeletal dynamics in cells.

Actins↗
PubMed1999

Neuronal differentiation and patterning in Xenopus: the role of cdk5 and a novel activator xp35.2.

Cdk5, a member of the cyclin-dependent kinase family, has been shown to play an important role in development of the central nervous system in mammals when partnered by its activator p35. Here we describe the cloning and characterization of a novel activator of cdk5 in Xenopus, Xp35.2. Xp35.2 is expressed during development initially in the earliest differentiating primary neurons in the neural plate and then later in differentiating neural tissue of the brain. This is in contrast to the previously described Xenopus cdk5 activator Xp35.1 which is expressed over the entire expanse of the neural plate in both proliferating and differentiating cells. Expression of both Xp35.1 and Xp35.2 and activation of cdk5 kinase occur when terminal neural differentiation is induced by neurogenin and neuro D overexpression but not when only early stages of neural differentiation are induced by noggin. Moreover, blocking cdk5 kinase activity specifically results in disruption and reduction of the embryonic eye where cdk5 and its Xp35 activators are expressed. Thus, cdk5/p35 complexes function in aspects of neural differentiation and patterning in the early embryo and particularly in formation of the eye.

Amino Acid Sequence↗
PubMed1999

Sequence analysis and expression of a cDNA clone encoding a 98-kDa allergen in Dermatophagoides farinae.

BACKGROUND: The important dust mite allergens identified to date are of molecular weights ranging from 14 to 60 kDa. Our previous protein study indicated that the 98-kDa native paramyosin in Dermatophagoides farinae mite showed IgE reactivity with 82% of the mite-sensitive asthmatic patients suggesting that it is a novel major mite allergen. This study described the isolation and characterization of the cDNA clone encoding the 98-kDa mite allergen. METHODS: A Dermatophagoides farinae cDNA library was constructed in lambda ZAPII vector and the library was immunoscreened with a monoclonal antibody 642. The cDNA insert was sub-cloned into M13 sequencing vector for single-stranded sequencing. The whole cDNA insert was expressed in pGEX-2T Escherichia coli expression system as a fusion protein with GST. The allergenicity of the recombinant peptides was tested by skin tests and IgE immunoassay. The IgE and IgG immunoassays were performed with sera from 20 mite-allergic patients. RESULTS: The cDNA clone Df642 was 2134 bp long, coding for a polypeptide of 711 amino acid residues. Protein sequence analysis and alignment confirmed that the deduced polypeptide is a mite paramyosin which is truncated slightly at the N- and C-terminuses. In vivo skin tests and in vitro IgE-binding study showed that 62% (13/21) and 50% (10/20) of the mite-sensitive asthmatic patients reacted positively with the recombinant Dermatophagoides farinae paramyosin, respectively. CONCLUSION: The study indicated that 98-kDa mite paramyosin is an important allergen.

Adolescent↗
PubMed1999

Is megalencephaly specific to autism?

Several recent reports have described the presence of increased head circumference (megalencephaly) in patients with autism. Although some studies have described reports of megalencephaly in other disorders such as schizophrenia in adults, few such studies have been performed in children and adolescents. In the present study, the authors compared 20 subjects with autism/ pervasive developmental disorder (DSM-IV; all males; mean age = 10.9 years) with 20 controls with attention deficit hyperactivity disorder (DSM-IV; all males; mean age = 11.1 years). Four subjects and five controls had evidence of megalencephaly. In addition to their core symptoms, the autistic subjects with megalencephaly were hyperactive and impulsive. These findings suggest that megalencephaly may not be specific to autism, and when present, it may index the presence of additional symptoms such as hyperactivity and impulsivity.

Adolescent↗
PubMed1998

Structural characterization and immunochemical detection of a fluorophore derived from 4-hydroxy-2-nonenal and lysine.

Aging and the progression of certain degenerative diseases are accompanied by increases in intracellular fluorescent material, termed "lipofuscin" and ceroid, respectively. These pigments are observed within granules composed, in part, of damaged protein and lipid. Modification of various biomolecules by aldehyde products of lipid peroxidation is believed to contribute to lipofuscin and ceroid formation. However, little direct evidence currently exists because the structures responsible for the fluorescent, cross-linked nature of this material are not well characterized. In this study, we have identified a fluorescent product formed in the reaction of Nalpha-acetyllysine and 4-hydroxy-2-nonenal (HNE), a major product of lipid peroxidation and the most reactive of these compounds under physiological conditions [Esterbauer, H., Shaur, R. J. & Zollner, H. (1991) Free Radical Biol. Med. 11, 81-128]. This fluorescent compound, characterized as a 2-hydroxy-3-imino-1,2-dihydropyrrol derivative, appears to form upon oxidative cyclization of the nonfluorescent 2:1 lysine-HNE Michael adduct-Schiff base cross-link. Polyclonal antibody was raised to the Nalpha-acetyllysine-HNE fluorophore and found to be highly specific to the chromophore structure of the compound. This antibody has been used to conclusively demonstrate that the lysine-HNE derivative of this fluorophore forms on protein upon exposure to HNE. The results of this study therefore provide the basis for future investigations on the contribution(s) of HNE-derived fluorophore formation to lipofuscin and ceroid accumulation.

Aldehydes↗
PubMed1998

Site-specific inactivation of aldose reductase by 4-hydroxynonenal.

Bovine lens aldose reductase (ALR2), which catalyzes the NADPH-dependent reduction of 4-hydroxy-2-nonenal (HNE), is readily inactivated by its own substrate in a time- and concentration-dependent manner. Both DTT and NADP+ can prevent enzyme inactivation but neither extensive dialysis nor thiol-reducing treatment were able to restore enzyme activity once inactivation had occurred. Unlike the native enzyme, S-glutathionyl-modified ALR2 is unaffected by HNE, and can be easily reverted to the native form under thiol-reducing conditions. Evidence is presented of the involvement of Cys298 in the inactivation process. Zofenoprilat, an antioxidant thiol compound, mimics the effect of GSH. The possibility is raised that enzyme thiolation may function as a protection mechanism against the irreversible modification of ALR2.

Aldehyde Reductase↗
PubMed1998

Human granulosa cells use high density lipoprotein cholesterol for steroidogenesis.

This study examines the ability of human high density lipoproteins (HDL3) to deliver cholesteryl esters to human granulosa cells and describes the selective cholesterol pathway by which this occurs. Luteinized cells obtained from subjects undergoing in vitro fertilization-embryo transfer procedures were incubated with native HDL3 (or radiolabeled or fluorescently labeled HDL cholesteryl esters) to determine whether cells from humans (in which HDL is not the primary circulating lipoprotein species) can nevertheless interiorize and appropriately process cholesteryl esters for steroidogenesis. The results indicate that hormone-stimulated granulosa cells actively and efficiently use human HDL-derived cholesterol for progesterone production. More than 95% of the mass of HDL cholesteryl esters entering cells does so through the nonlysosomal (selective) pathway, i.e. cholesteryl esters released from HDL are taken up directly by the cells without internalization of apoproteins. Once internalized, the cholesteryl esters are either hydrolyzed and directly used for steroidogenesis or stored in the cells as cholesteryl esters until needed. The utilization of the internalized cholesteryl esters is a hormone-regulated event; i.e. luteinized human granulosa cells internalize and store large quantities of HDL-donated cholesteryl esters when available, but further processing of the cholesteryl esters (hydrolysis, re-esterification, or use in steroidogenesis) does not occur unless the cells are further stimulated to increase progesterone secretion.

Boron Compounds↗
PubMed1997

Genghis Khan (Gek) as a putative effector for Drosophila Cdc42 and regulator of actin polymerization.

The small GTPases Cdc42 and Rac regulate a variety of biological processes, including actin polymerization, cell proliferation, and JNK/mitogen-activated protein kinase activation, conceivably via distinct effectors. Whereas the effector for mitogen-activated protein kinase activation appears to be p65PAK, the identity of effector(s) for actin polymerization remains unclear. We have found a putative effector for Drosophila Cdc42, Genghis Khan (Gek), which binds to Dcdc42 in a GTP-dependent and effector domain-dependent manner. Gek contains a predicted serine/threonine kinase catalytic domain that is 63% identical to human myotonic dystrophy protein kinase and has protein kinase activities. It also possesses a large coiled-coil domain, a putative phorbol ester binding domain, a pleckstrin homology domain, and a Cdc42 binding consensus sequence that is required for its binding to Dcdc42. To study the in vivo function of gek, we generated mutations in the Drosophila gek locus. Egg chambers homozygous for gek mutations exhibit abnormal accumulation of F-actin and are defective in producing fertilized eggs. These phenotypes can be rescued by a wild-type gek transgene. Our results suggest that this multidomain protein kinase is an effector for the regulation of actin polymerization by Cdc42.

Actins↗
PubMed1996

Intracellular events in the "selective" transport of lipoprotein-derived cholesteryl esters.

The current study utilizes human, apoE-free high density lipoprotein reconstituted with a highly specific fluorescent-cholesteryl ester probe to define the initial steps and regulatory sites associated with the "selective" uptake and intracellular itinerary of lipoprotein-derived cholesteryl esters. Bt2cAMP-stimulated ovarian granulosa cells were used as the experimental model, and both morphological and biochemical fluorescence data were obtained. The data show that cholesteryl ester provided through the selective pathway is a process which begins with a temperature-independent transfer of cholesteryl ester to the cell's plasma membrane. Thereafter transfer of the lipid proceeds rapidly and accumulates prominently in a perinuclear region (presumed to be the Golgi/membrane sorting compartment) and in lipid storage droplets of the cells. The data suggest that lipid transfer proteins (or other small soluble proteins) are not required for the intracellular transport of the cholesteryl esters, nor is an intact Golgi complex or an intact cell cytoskeleton (although the transfer is less efficient in the presence of certain microtubule-disrupting agents). The intracellular transfer of the cholesteryl esters is also somewhat dependent on an energy source in that a glucose-deficient culture medium or a combination of metabolic inhibitors reduces the efficiency of the transfer. A protein-mediated event may be required for cholesteryl ester internalization from the plasma membrane, in that N-ethylmaleimide dramatically blocks the internalization phase of the selective uptake process. Taken together these data suggest that the selective pathway is a factor-dependent, energy-requiring cholesteryl ester transport system, in which lipoprotein-donated cholesteryl esters probably flow through vesicles or intracellular membrane sheets and their connections, rather than through the cell cytosol.

Adenosine Triphosphatases↗
PubMed1996

Chemical characterization of a protein-4-hydroxy-2-nonenal cross-link: immunochemical detection in mitochondria exposed to oxidative stress.

We have previously shown that incubation of the model protein glucose-6-phosphate dehydrogenase (Glu-6-PDH) from the bacterium Leuconostoc mesenteroides with 4-hydroxy-2-nonenal (HNE), a major product of lipid peroxidation, results in the formation of cross-linked protein. HNE-modified protein is resistant to proteolytic degradation and acts as an inhibitor of the multicatalytic proteinase. It was therefore important to establish the chemistry of the cross-linking reaction. The formation of cross-linked Glu-6-PDH is associated with the nearly exclusive loss of lysine residues. For this reason the reaction of N-acetyllysine with HNE has been investigated. The epsilon-amino group of lysine reacts with the double bond (C3) and the carbonyl (C1) functions of HNE via Michael addition and Schiff base formation resulting in the production of a 2:1 amino acid-HNE cross-link. Chromatographic detection of this adduct in the acid hydrolysate of HNE-treated Glu-6-PDH reveals that this chemistry is responsible for the formation of cross-linked protein. Antibody to the reduced form of the 2:1 lysine-HNE adduct was prepared. The antibody was used to demonstrate that exposure of isolated liver mitochondria to oxidative stress led to the formation of intra- and intermolecular protein-HNE cross-links. The results of the present study indicate that modifications to protein by lipid peroxidation products may be physiologically relevant and could contribute to the disease- and age-related buildup of damaged protein.

Aldehydes↗
PubMed1996

Dietary exposures to selected metals and pesticides.

Average daily dietary exposures to 11 contaminants were estimated for approximately 120,000 U.S. adults by combining data on annual diet, as measured by a food frequency questionnaire, with contaminant residue data for table-ready foods that were collected as part of the annual U.S. Food and Drug Administration Total Diet Study. The contaminants included in the analysis were four heavy metals (arsenic, cadmium, lead, mercury), three organophosphate pesticides (chlorpyrifos, diazinon, malathion), and four organochlorine pesticides (dieldrin, p,p'-DDE, lindane, heptachlor epoxide). Dietary exposures to these contaminants were highly variable among individuals, spanning two to three orders of magnitude. Intraindividual exposures to the metals, organophosphates, and organochlorines were estimated to be strongly correlated; Pearson's correlation coefficients ranged from 0.28 for lindane:dieldrin to 0.84 for lead:mercury. For some of the compounds (e.g., arsenic and dieldrin), a substantial fraction of the population was estimated to have dietary intakes in excess of health-based standards established by the EPA. Before use for risk assessment or epidemiologic purposes, however, the validity of the exposure estimates must be evaluated by comparison with biological indicators of chronic exposure. Because of their low detection rate in table-ready foods, the estimated distributions of exposures for dieldrin, p,p'-DDE, heptachlor epoxide, lindane, diazinon, and chlorpyrifos were found to be sensitive to assumed values for nondetect samples. Reliable estimates of the population distribution of dietary exposures to most other contaminants cannot be made currently, due to their low rate of detection in table-ready foods. Monitoring programs that use more sensitive study designs and population-based assessments for other subpopulations should be a priority for future research.

Adult↗
PubMed1995

Brief report: thought disorder in Asperger syndrome: comparison with high-functioning autism.

Asperger syndrome (AS) is a pervasive developmental disorder generally regarded as a variant of autism. While it has been included in the ICD-10 and DSM-IV as a distinct diagnostic entity, it is still unclear to what extent it differs from high-functioning autism (HFA). Persons with HFA have been reported to show a variety of deficits of thought processes. Abnormalities such as poor reality testing, perceptual distortions, and areas of cognitive slippage have been described using the Rorschach inkblot test (Dykens, Volkmar, & Glick, 1991). Since AS has been conceptualized as a mild variant of autism, we hypothesized that persons with AS will have fewer abnormalities on the Rorschach test compared to persons with HFA. To test this hypothesis, we compared 12 subjects with AS (ICD-10, 10 male, mean age = 12.2 +/- 3.3 years, mean full-scale IQ = 99.6) with 8 subjects with HFA (ICD-10/DSM-III-R, 7 male, mean age = 12.2 +/- 3.8 years, mean full-scale IQ = 83.4) on the Rorschach test. AS subjects demonstrated a trend towards greater levels of disorganized thinking than the HFA group. They were also more likely to be classified as "Introversive" suggesting that AS subjects may have more complex inner lives involving elaborate fantasies, Also, AS subjects tended to be more focused on their internal experiences. However, overall, the Rorschach test was not found to differentiate the two diagnostic groups on the majority of structural variables. Implications of these findings are discussed with regard to the diagnostic validity of Asperger syndrome.

Adolescent↗
PubMed1995

The reaction of 4-hydroxy-2-nonenal with N alpha-acetyl-L-histidine.

The reaction of 4-hydroxy-2-nonenal and N alpha-acetyl-L-histidine was studied in pH 7.1 phosphate buffer. The main product isolated was assigned a cyclic hemiacetal structure formed by the addition of one of the imidazole nitrogen atoms to the alpha,beta-unsaturated aldehyde system of 4-hydroxy-2-nonenal. This structural assignment was based on the analyses of the NMR and mass spectral data of two derivatives obtained from the cyclic hemiacetal. The establishment of this cyclic hemiacetal structure supports the proposal made by Uchida and Stadtman6 that 4-hydroxy-2-nonenal modified histidyl residues in insulin by a Michael reaction.

Aldehydes↗
PubMed1995

Obstetric factors in Asperger syndrome: comparison with high-functioning autism.

Asperger syndrome (AS) is a pervasive developmental disorder widely regarded as a mild variant of autism. To investigate if AS is associated with a history of fewer obstetric insults compared to autism, we examined the developmental history and obstetric records of 10 males with AS (mean full scale IQ 95.3), and compared them with 10 autistic males with a full scale IQ of 70 or above (so-called high-functioning autism; mean full scale IQ 82.6). Males with AS showed a trend toward lower Apgar scores at one minute (chi-square = 4; df = 1; P = 0.04) and were more likely to have been born to mothers outside the optimal age group of 20-30 years (chi-square = 5; df = 1; P = 0.02). They were also less likely to have been irritable and floppy as infants (chi-square = 3.8; df = 1; P = 0.05). However, the total optimality scores did not differ significantly between the two groups.

Adolescent↗