Search PubMed⌕ Search

Biomedical subjects

R Li

Publications and source records attributed to R Li.

At least 433 records · Page 24Linked to original sources
PubMed1995

The three-dimensional structure of NAD(P)H:quinone reductase, a flavoprotein involved in cancer chemoprotection and chemotherapy: mechanism of the two-electron reduction.

Quinone reductase [NAD(P)H:(quinone acceptor) oxidoreductase, EC 1.6.99.2], also called DT diaphorase, is a homodimeric FAD-containing enzyme that catalyzes obligatory NAD(P)H-dependent two-electron reductions of quinones and protects cells against the toxic and neoplastic effects of free radicals and reactive oxygen species arising from one-electron reductions. These two-electron reductions participate in the reductive bioactivation of cancer chemotherapeutic agents such as mitomycin C in tumor cells. Thus, surprisingly, the same enzymatic reaction that protects normal cells activates cytotoxic drugs used in cancer chemotherapy. The 2.1-A crystal structure of rat liver quinone reductase reveals that the folding of a portion of each monomer is similar to that of flavodoxin, a bacterial FMN-containing protein. Two additional portions of the polypeptide chains are involved in dimerization and in formation of the two identical catalytic sites to which both monomers contribute. The crystallographic structures of two FAD-containing enzyme complexes (one containing NADP+, the other containing duroquinone) suggest that direct hydride transfers from NAD(P)H to FAD and from FADH2 to the quinone [which occupies the site vacated by NAD(P)H] provide a simple rationale for the obligatory two-electron reductions involving a ping-pong mechanism.

Amino Acid Sequence↗
PubMed1995

A spectrophotometric determination of alpha-dicarbonyl compounds and its application to the enzymatic formation of alpha-ketobutyrate.

A simple, rapid, and sensitive spectrophotometric method has been developed for the determination of alpha-keto acids, alpha-diketones, and alpha-ketoaldehydes using o-phenylenediamine as a derivatizing reagent in acidic media to yield chromophoric quinoxaline derivatives. Application to the determination of alpha-ketobutyrate and the rate of its formation from isomerization of vinylglycolate catalyzed by mandelate racemase is described. Under optimal conditions 1 to 4 mM o-phenylenediamine in 80% acetic acid was incubated at 50 degrees C for 10 min with the alpha-dicarbonyl compounds. Solutions of alpha-ketobutyrate as low as 5 microM in the buffer solution can be measured quantitatively. This method has also been used for determining the rate of formation of the phenylglyoxalate from (S)-mandelate catalyzed by (S)-mandelate dehydrogenase.

Butyrates↗
PubMed1995

Mice lacking the c-rel proto-oncogene exhibit defects in lymphocyte proliferation, humoral immunity, and interleukin-2 expression.

The c-rel proto-oncogene, which is expressed predominantly in hemopoietic cells encodes a subunit of the NF-kappa B-like family of transcription factors. In mice with an inactivated c-rel gene, whereas development of cells from all hemopoietic lineages appeared normal, humoral immunity was impaired and mature B and T cells were found to be unresponsive to most mitogenic stimuli. Phorbol ester and calcium ionophore costimulation, in contrast to certain membrane receptor-mediated signals, overcame the T cell-proliferative defect, demonstrating that T cell proliferation occurs by Rel-dependent and -independent mechanisms. The ability of exogenous interleukin-2 to restore T Cell, but not B cell, proliferation indicates that Rel regulates the expression of different genes in B and T cells that are crucial for cell division and immune function.

Aging↗
PubMed1995

Human THP-1 monocyte-macrophage membrane binding proteins: distinct receptor(s) for triglyceride-rich lipoproteins.

An apolipoprotein (apo) E- and lipoprotein lipase-independent, high affinity, saturable and specific binding site and pathway for uptake of certain triglyceride-rich lipoproteins (TGRLP) by human monocyte-macrophages that leads to lipid accumulation and foam cell formation in vitro has been reported; two membrane binding activities were identified as receptor candidates with apparent molecular masses of 200 and 235 kDa [Gianturco et al. (1994) J. Lipid Res. 35, 1674-1687]. Here we present new evidence that these activities are TGRLP receptors with unique biochemical properties which distinguish them from other lipoprotein receptors. Protease and heparinase susceptibility studies demonstrate that (1) these activities have essential protein, but not heparan sulfate proteoglycan (HSPG) components; (2) the membrane binding proteins (MBPs) are located on the cell surface; (3) HSPGs do not facilitate TGRLP binding to this specific cellular site. Upon reduction, MBP 200 and 235 are both converted into a single, new binding activity of intermediate mobility (MBP 200R); all MBP forms displayed high affinity, saturable TGRLP binding with similar Kds (1.4-2.2 micrograms/mL). Notably, MBP 200R retained the combined ligand binding capacity of MBP 200 and 235 prior to reduction, demonstrating that, unlike members of the LDL receptor or the scavenger receptor families, disulfide bonds are not critical for activity. At 65 degrees C, MBP 235 was converted into MBP 200 without loss of total binding activity, suggesting heat dissociates a small subunit not required for binding from a common large protein subunit that binds TGRLP. Since the MBPs are found on the cell surface, are themselves functionally and structurally related, have distinctly different biochemical properties from members of the LDL receptor and scavenger receptor families, and share all critical characteristics with the cellular binding site, we hypothesize that they represent a new and unique receptor family for apoE- and lipoprotein lipase-independent uptake of TGRLP by human monocyte-macrophages.

Apolipoproteins E↗
PubMed1995

Absence of yolk sac hematopoiesis from mice with a targeted disruption of the scl gene.

The scl gene encodes a basic-helix-loop-helix transcription factor which was identified through its involvement in chromosomal translocations in T-cell leukemia. To elucidate its physiological role, scl was targeted in embryonic stem cells. Mice heterozygous for the scl null mutation were intercrossed and their offspring were genotyped. Homozygous mutant (scl-/-) pups were not detected in newborn litters, and analysis at earlier time points demonstrated that scl-/- embryos were dying around embryonic day 9.5. The scl-/- embryos were pale, edematous, and markedly growth retarded after embryonic day 8.75. Histological studies showed complete absence of recognizable hematopoiesis in the yolk sac of these embryos. Early organogenesis appeared to be otherwise normal. Culture of yolk sac cells of wild-type, heterozygous, and homozygous littermates confirmed the absence of hematopoietic cells in scl-/- yolk sacs. Reverse transcription PCR was used to examine the transcripts of several genes implicated in early hematopoiesis. Transcripts of GATA-1 and PU.1 transcription factors were absent from RNA from scl-/- yolk sacs and embryos. These results implicate scl as a crucial regulator of early hematopoiesis.

Animals↗
PubMed1995

Myogenic and morphogenetic defects in the heart tubes of murine embryos lacking the homeo box gene Nkx2-5.

The murine homeo box gene Nkx2-5 is expressed in precardiac mesoderm and in the myocardium of embryonic and fetal hearts. Targeted interruption of Nkx2-5 resulted in abnormal heart morphogenesis, growth retardation and embryonic lethality at approximately 9-10 days postcoitum (p.c.). Heart tube formation occurred normally in mutant embryos, but looping morphogenesis, a critical determinant of heart form, was not initiated at the linear heart tube stage (8.25-8.5 days p.c.). Commitment to the cardiac muscle lineage, expression of most myofilament genes and myofibrillogenesis were not compromised. However, the myosin light-chain 2V gene (MLC2V) was not expressed in mutant hearts nor in mutant ES cell-derived cardiocytes. MLC2V expression normally occurs only in ventricular cells and is the earliest known molecular marker of ventricular differentiation. The regional expression in mutant hearts of two other ventricular markers, myosin heavy-chain beta and cyclin D2, indicated that not all ventricle-specific gene expression is dependent on Nkx2-5. The data demonstrate that Nkx2-5 is essential for normal heart morphogenesis, myogenesis, and function. Furthermore, this gene is a component of a genetic pathway required for myogenic specialization of the ventricles.

Animals↗
PubMed1995

The role of thyroid hormone and promoter diversity in the regulation of nuclear encoded mitochondrial proteins.

Thyroid hormone regulates the in vivo expression of a selected set of rat nuclear genes encoding mitochondrial inner membrane proteins. Certain mRNAs, such as that for cytochrome c1, are increased as much as 20-50-fold, while others, such as core protein 1 of Complex III and the F1-ATPase beta-subunit do not respond. The promoter region of human cytochrome c1 also supports thyroid hormone induction of a reporter gene in transient transfection experiments. Thus, thyroid hormone regulates only selected genes, even for subunits within the same complex and in widely varying species. By contrast, growth activation of quiescent NIH3T3 cells, a second paradigm used for stimulating mitochondrial biogenesis, does not increase cytochrome c1 mRNA but does increase F1-ATPase beta-subunit mRNA. These findings suggest that nuclear OXPHOS genes are not necessarily expressed in a coordinated manner, and that multiple regulatory circuits might exist which are linked to different physiological stimuli. Analysis of the promoters of several OXPHOS genes reveals a great diversity and heterogeneity of transfactor binding elements. No single regulatory feature exists which could account for a coordinated expression of all OXPHOS genes. The potential diversity for regulating expression of nuclear OXPHOS genes raises the possibility for the existence of disease states linked to regulatory defects.

3T3 Cells↗
PubMed1995

Purification of the human THP-1 monocyte-macrophage triglyceride-rich lipoprotein receptor.

Previously we reported that human blood-borne and THP-1 monocyte-macrophages have an apolipoprotein E- and lipoprotein lipase-independent, high affinity, specific binding site for the uptake and degradation of hypertriglyceridemic VLDL and plasma chylomicrons distinct from the LDL receptor gene family and the acetyl LDL receptor (Gianturco et al., J. Lipid Res. 35:1674-1687, 1994). Ligand blot analyses identified two cell-surface, structurally related membrane binding proteins as receptor candidates of M(r) approximately 200 kDa and M(r) approximately 235 kDa which are converted into a single ligand binding species of intermediate mobility upon reduction. We now report a approximately 1200-fold purification of the reduced candidate receptor protein from cultured THP-1 monocytes.

Chromatography, DEAE-Cellulose↗
PubMed1995

Immunosuppressive activity of chemically synthesized gangliosides.

New chemical synthetic methods have permitted the synthesis of a spectrum of glycosphingolipid molecular species, some of which are not naturally occurring. Here we have studied a number of chemically synthesized gangliosides for immunosuppressive activity, using a human in vitro specific antigen (tetanus toxoid)-induced assay of the cellular immune response. Chemically synthesized GM3 and GM4 had the same high degree of immunosuppressive activity as did natural GM3 and GM4 gangliosides, verifying that inhibition is intrinsic to the ganglioside molecules and not caused by other molecules sometimes found in natural preparations (e.g., proteins). Studies of modified molecular species of GM3 and GM4, also prepared by chemical synthesis, have shown the influence of certain structural details upon the immunosuppressive activity of gangliosides: (i) the inverse relationship between fatty acyl chain length and immunosuppressive activity is extended to even shorter chain lengths, with the synthetic gangliosides d18:1-C2:0-GM3 and d18:1-C14:0-GM3 being more immunosuppressive than d18:1-C18:0-GM3 and d18:1-C24:0-GM3; (ii) hydroxylation of the fatty acyl group decreases immunosuppressive activity; (iii) substitution of an S-glycosidic bond for an O-glycosidic bond in the sialic acid ketosidic linkage in GM4 does not alter its activity; and (iv) modifications of the sialic acid group variably influence immunosuppressive activity, since KDN-GM3 and -GM4 ganglioside analogues, which contain a 3-deoxy-D-glycero-D-galacto-2-nonulopyranosonic acid in place of N-acetylneuraminic acid, retain activity, while other modifications such as 8-epi-GM3, and to a lesser extent 9-deoxy-GM3, reduce immunosuppressive activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Brain Chemistry↗
PubMed1995

Human tumor gangliosides inhibit murine immune responses in vivo.

Gangliosides which are shed by tumor cells clearly inhibit cellular immune responses in vitro. However, the immunosuppressive activity of these molecules have been more difficult to ascertain in vivo. Here we have adapted a murine model to determine the effects of tumor gangliosides in an in vivo microenvironment, the lymph node draining the site of stimulation by allogeneic cells. In this model, allogeneic splenocytes (BALB/c) are s.c. injected into C3H mice. The cellular immune response in the draining popliteal lymph nodes 4 days later is evidenced as an increase in lymph node mass (2-fold), lymphocyte number (6-fold), and lymphocyte DNA synthesis (6-fold). Purified human neuroblastoma gangliosides (10 nmol) coinjected with the stimulating allogeneic cells significantly suppressed this in vivo immune response. The increase in the lymph node mass was reduced by 65% (0.66 versus 1.89 mg), the increase in lymphocyte number (4.0 x 10(6) cells/node) was almost completely inhibited (1.1 x 10(6) cells/node), and in vitro [3H]thymidine uptake by the lymphocytes recovered in vivo was reduced by 80%. In contrast to the inhibition by tumor gangliosides, liposomes of cholesterol:lecithin were not inhibitory. Thus, tumor gangliosides, specifically, modulate cellular immune responses in vivo, which may contribute to the observed enhancement of tumor formation by these molecules.

Animals↗
PubMed1995

Tb3+ binding to human erythrocyte spectrin resulting in conformation change and aggregation.

The Tb3+ binding to spectrin tetramer (SPT) was studied by Tb3+ fluorescence titration and CD spectra. The results indicated that the total high-affinity Tb3+ binding sites are n1 = 330, with average Kd = 3.6 x 10(-6) M. Among them, ca. 90 sites are of higher affinity and are probably more specific to Tb3+ than the remaining sites. There are 520 low affinity Tb3+ binding sites with average Kd = 1.5 x 10(-5) M. Fluorescence and CD spectra revealed that the alpha-helix content of SPT decreased with Tb3+ binding to specific sites and further binding did not result in conformation change. Tb3+ binding to SPT and the subsequent reactions were studied by employing stopped-flow fluorescence and light scattering methods. The studies demonstrate that this is a multistep reaction assembly: high-affinity terbium binding-conformation change-aggregation-low-affinity terbium binding--the second conformation change. The critical Tb3+ concentration-induced spectrin dimer (SPD) aggregation was determined with a light scattering method.

Binding Sites↗
PubMed1995

An animal model for mania: preliminary results.

1. In human bipolar patients mania and bipolar depression are both characterized by decreased membrane Na,K-AtPase activity. Additionally, digoxin neurotoxicity in patients frequently presents with symptoms of mania or depression. 2. These findings suggest that central nervous system Na,K-ATPase inhibition may play a pathophysiologic role in bipolar illness. 3. The authors tested this hypothesis by administering intracerebroventricular (i.c.v.) ouabain to rats at sublethal doses. The authors then measured behavioral activity as total square crossings in an open field. 4. Motoric activity was significantly increased by i.c.v. administration of 5 microliters of ouabain at 10(-3) M. This preliminary study suggests that i.c.v. ouabain administration may provide a useful animal model of mania that is based on observed biochemical changes in humans.

Animals↗
PubMed1995

The role of neuropeptides in learning and memory: possible mechanisms.

Endogenous neuropeptides such as vasopressin, adrenocorticotropin and opioids have significant effects on learning and memory. However, because of the complexity of behaviour, that is defined as 'learning' and 'memory' and, because of the limitation of current knowledge, it has been difficult to interpret these behavioural data, especially via neural mechanisms. The application of modern experimental techniques including molecular biology such as cloning and electrophysiology, such as patch-clamp, has had a significant impact upon the concepts about drugs, including neuropeptides action sites. This allows us to try to interpret some behavioural consequences influenced by neuropeptides. The data on effects of some neuropeptides on behaviours and their possible mechanisms are reviewed. Whatever the mechanisms, vasopressin, adrenocorticotropic hormone and endogenous opioids seem to have important effects upon learning and memory and these open up the possibility that drugs enhance cognitive functions or treat dementia via alteration of functions of neuropeptides. Some criteria are proposed for evaluating the validity of behavioural tests for neuropeptides.

Adrenocorticotropic Hormone↗
PubMed1995

Relation of activity levels to body fat in infants 6 to 12 months of age.

We examined longitudinally the relation between body fatness and physical activity, adjusting for energy intake, in 31 healthy white infants. Measures of physical activity, dietary intake, and body composition were obtained at 6, 9, and 12 months of age. The percentage of body fat was inversely related to activity level, an association that became stronger with increasing age and remained significant after adjustment for dietary energy intake. The percentage of body fat was not related to energy consumed per lean body mass regardless of high or low activity level, nor was energy consumed related to physical activity. We conclude that the percentage of body fat in infants may be related more to their activity levels than to their energy intake.

Adipose Tissue↗
PubMed1995

Regulation of cortical actin cytoskeleton assembly during polarized cell growth in budding yeast.

We have established an in vitro assay for assembly of the cortical actin cytoskeleton of budding yeast cells. After permeabilization of yeast by a novel procedure designed to maintain the spatial organization of cellular constituents, exogenously added fluorescently labeled actin monomers assemble into distinct structures in a pattern that is similar to the cortical actin distribution in vivo. Actin assembly in the bud of small-budded cells requires a nucleation activity provided by protein factors that appear to be distinct from the barbed ends of endogenous actin filaments. This nucleation activity is lost in cells that lack either Sla1 or Sla2, proteins previously implicated in cortical actin cytoskeleton function, suggesting a possible role for these proteins in the nucleation reaction. The rate and the extent of actin assembly in the bud are increased in permeabilized delta cap2 cells, providing evidence that capping protein regulates the ability of the barbed ends of actin filaments to grow in yeast cells. Actin incorporation in the bud can be stimulated by treating the permeabilized cells with GTP-gamma S, and, significantly, the stimulatory effect is eliminated by a mutation in CDC42, a gene that encodes a Rho-like GTP-binding protein required for bud formation. Furthermore, the lack of actin nucleation activity in the cdc42 mutant can be complemented in vitro by a constitutively active Cdc42 protein. These results suggest that Cdc42 is closely involved in regulating actin assembly during polarized cell growth.

Actins↗