Self-assembly of unlike homopolymers into hollow spheres in nonselective solvent.
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Chloroform↗
Biomedical subjects
M Jiang
Publications and source records attributed to M Jiang.
GABAergic neurons that pioneer hippocampal area CA1 of the mouse: morphologic features and multiple fates.
Dramatic changes occur in the expression of glutamic acid decarboxylase (GAD67) immunoreactivity in mouse hippocampus during postnatal development. Most striking is the presence of a dense population of immunopositive cells in stratum radiatum and stratum oriens in area CA1 during the first postnatal week. Between days 5 and 10, these cells disappear and the GAD67 immunoreactivity begins to resemble that of adulthood. These neurons are considered pioneer cells, and studies were undertaken to determine their fate. Between days 5 and 50, area CA1 doubles in size; however, the loss of cells expressing GAD67 mRNA cannot be explained solely by dilution resulting from hippocampal growth. In stratum radiatum, cell loss is particularly dramatic. Although between days 5 and 15, many cells seem to migrate from stratum radiatum to its border with stratum lacunosum-moleculare, both fate maps of pioneer cells labeled with bromodeoxyuridine (BrdU) on embryonic day 13 (E13) and in situ DNA end-labeling studies suggest that some cells die by means of programmed cell death. However, not all pioneer cells die, because many cells labeled with BrdU on E13 are present in adulthood and express markers for and have anatomic features of hippocampal interneurons. In conclusion, events that underlie the age-dependent disappearance of gamma-aminobutyric acid (GABA) -ergic pioneer cells are complex and cannot be completely explained by dilution in an expanding neuropile. Although some GABAergic pioneer cells likely undergo programmed cell death during the first postnatal weeks, others relocate within hippocampal laminae and terminally differentiate into the interneurons of adulthood.
Aging↗
Alternatively folded choriogonadotropin analogs. Implications for hormone folding and biological activity.
Most heterodimeric proteins are stabilized by intersubunit contacts or disulfide bonds. In contrast, human chorionic gonadotropin (hCG) and other glycoprotein hormones are secured by a strand of their beta-subunits that is wrapped around alpha-subunit loop 2 "like a seatbelt." During studies of hCG synthesis in COS-7 cells, we found that, when the seatbelt was prevented from forming the disulfide that normally "latches" it to the beta-subunit, its carboxyl-terminal end can "scan" the surface of the heterodimer and become latched by a disulfide to cysteines substituted for residues in the alpha-subunit. Analogs in which the seatbelt was latched to residues 35, 37, 41-43, and 56 of alpha-subunit loop 2 had similar lutropin activities to those of hCG; that in which it was latched to residue 92 at the carboxyl terminus had 10-20% the activity of hCG. Attachment of the seatbelt to alpha-subunit residues 45-51, 86, 88, 90, and 91 reduced lutropin activity substantially. These findings show that the heterodimer can form before the beta-subunit has folded completely and support the notions that the carboxyl-terminal end of the seatbelt, portions of alpha-subunit loop 2, and the end of the alpha-subunit carboxyl terminus do not participate in lutropin receptor interactions. They suggest also that several different architectures could have been sampled without disrupting hormone activity as the glycoprotein hormones diverged from other cysteine knot proteins.
Alanine↗
Synthesis and biological evaluation of clitocine analogues as adenosine kinase inhibitors.
Adenosine kinase (AK) is the primary enzyme responsible for adenosine metabolism. Inhibition of AK effectively increases extracellular adenosine concentrations and represents an alternative approach to enhance the beneficial actions of adenosine as compared to direct-acting receptor agonists. Clitocine (3), isolated from the mushroom Clitocybe inversa, has been found to be a weak inhibitor of AK. We have prepared a number of analogues of clitocine in order to improve its potency and demonstrated that 5'-deoxy-5'-amino-clitocine (7) improved AK inhibitory potency by 50-fold.
Adenosine Kinase↗
A gene expression map for Caenorhabditis elegans.
We have assembled data from Caenorhabditis elegans DNA microarray experiments involving many growth conditions, developmental stages, and varieties of mutants. Co-regulated genes were grouped together and visualized in a three-dimensional expression map that displays correlations of gene expression profiles as distances in two dimensions and gene density in the third dimension. The gene expression map can be used as a gene discovery tool to identify genes that are co-regulated with known sets of genes (such as heat shock, growth control genes, germ line genes, and so forth) or to uncover previously unknown genetic functions (such as genomic instability in males and sperm caused by specific transposons).
Algorithms↗
p53 binds the nuclear matrix in normal cells: binding involves the proline-rich domain of p53 and increases following genotoxic stress.
The tumour suppressor p53 is a multifunctional protein important for the maintenance of genomic integrity. It is able to form molecular complexes with different DNA targets and also with cellular proteins involved in DNA transcription and DNA repair. In mammalian cells the biochemical processing of DNA occurs on a nuclear sub-structure termed the nuclear matrix. Previously Deppert and co-workers have identified p53 in association with the nuclear matrix in viral- and non-viral transformed cell lines. In the present study we demonstrate, for the first time, that p53 is bound to the nuclear matrix in primary cultures of normal mammalian cells and that this binding increases following DNA damage. Analysis of cell lines expressing structural mutants of p53 revealed that association with the nuclear matrix is independent of the tertiary and quaternary structure of p53. However, the proline-rich domain towards the N-terminus of p53 (residues 67 to 98) appeared important for binding to the nuclear matrix. This was demonstrated by TET-ON regulated expression of p53-derived constructs in p53(-/-) murine embryonic fibroblasts (MEF p53(-/-)). The proline-rich domain of p53 has potential for SH3 protein-protein interaction, and has a role in p53-mediated apoptosis and possibly base excision repair of DNA damage. We discuss our observations in relation to the ability of p53 to facilitate DNA repair and also review evidence indicating that matrix-bound p53 in SV40-transformed cells may facilitate the transforming potential of SV40 large T antigen.
Animals↗
Interrupting the template strand of the T7 promoter facilitates translocation of the DNA during initiation, reducing transcript slippage and the release of abortive products.
We have explored the effects of a variety of structural and sequence changes in the initiation region of the phage T7 promoter on promoter function. At promoters in which the template strand (T strand) is intact, initiation is directed a minimal distance of 5 nt downstream from the binding region. Although the sequence of the DNA surrounding the start site is not critical for correct initiation, it is important for melting of the promoter and stabilization of the initiation complex. At promoters in which the integrity of T strand is interrupted by nicks or gaps between -5 and -2 the enzyme continues to initiate predominately at +1. However, under these conditions there is a decrease in the release of abortive products of 8-10 nt, a decrease in the synthesis of poly(G) products (which arise by slippage of the nascent transcript), and a defect in displacement of the RNA. We propose that unlinking the binding and initiation regions of the T strand changes the manner in which this strand is retained in the abortive complex, reducing or eliminating the need to pack or "scrunch" the strand into the complex during initiation and lowering a thermodynamic barrier to its translocation.
Bacteriophage T7↗
Discovery of 4-amino-5-(3-bromophenyl)-7-(6-morpholino-pyridin-3-yl)pyrido[2,3-d]pyrimidine, an orally active, non-nucleoside adenosine kinase inhibitor..
Adenosine (ADO) is an endogenous homeostatic inhibitory neuromodulator that reduces cellular excitability at sites of tissue injury and inflammation. Inhibition of adenosine kinase (AK), the primary metabolic enzyme for ADO, selectively increases ADO concentrations at sites of tissue trauma and enhances the analgesic and antiinflammatory actions of ADO. Optimization of the high-throughput screening lead, 4-amino-7-aryl-substituted pteridine (5) (AK IC(50) = 440 nM), led to the identification of compound 21 (4-amino-5-(3-bromophenyl)-7-(6-morpholino-pyridin-3-yl)pyrido [2,3-d]pyrimidine, ABT-702), a novel, potent (AK IC(50) = 1.7 nM) non-nucleoside AK inhibitor with oral activity in animal models of pain and inflammation.
Adenosine Kinase↗
Dipyridamole inhibits sickling-induced cation fluxes in sickle red blood cells.
Sickling-induced cation fluxes contribute to cellular dehydration of sickle red blood cells (SS RBCs), which in turn potentiates sickling. This study examined the inhibition by dipyridamole of the sickling-induced fluxes of Na(+), K(+), and Ca(++) in vitro. At 2% hematocrit, 10 microM dipyridamole inhibited 65% of the increase in net fluxes of Na(+) and K(+) produced by deoxygenation of SS RBCs. Sickle-induced Ca(++) influx, assayed as (45)Ca(++) uptake in quin-2-loaded SS RBCs, was also partially blocked by dipyridamole, with a dose response similar to that of Na(+) and K(+) fluxes. In addition, dipyridamole inhibited the Ca(++)-activated K(+) flux (via the Gardos pathway) in SS RBCs, measured as net K(+) efflux in oxygenated cells exposed to ionophore A23187 in the presence of external Ca(++), but this effect resulted from reduced anion conductance, rather than from a direct effect on the K(+) channel. The degree of inhibition of sickling-induced fluxes was dependent on hematocrit, and up to 30% of dipyridamole was bound to RBC membranes at 2% hematocrit. RBC membrane content of dipyridamole was measured fluorometrically and correlated with sickling-induced flux inhibition at various concentrations of drug. Membrane drug content in patients taking dipyridamole for other clinical indications was similar to that producing inhibition of sickling-induced fluxes in vitro. These data suggest that dipyridamole might inhibit sickling-induced fluxes of Na(+), K(+), and Ca(++) in vivo and therefore have potential as a pharmacological agent to reduce SS RBC dehydration. (Blood. 2001;97:3976-3983)
Anemia, Sickle Cell↗
Increased oxidative stress in childhood atopic dermatitis.
Atopic dermatitis (AD) is a chronic inflammatory skin disease of unknown etiology. To examine the involvement of impaired homeostasis of oxygen/nitrogen radicals in childhood AD, we compared the levels of urinary 8-hydroxy-2'-deoxyguanosine (marker of oxidative stress), nitrite/nitrate (marker of nitric oxide synthesis) and selenium (marker of selenium store) in 27 children with AD to those of 25 healthy control children. Urinary 8-hydroxy-2'-deoxyguanosine was significantly higher and nitrite/nitrate levels were significantly lower in patients with AD than in the control. Urinary selenium levels were similar in both groups. Our findings suggest that impaired homeostasis of oxygen/nitrogen radicals and increased oxidative stress are involved in the pathophysiology of childhood AD, and indicate that suppression of oxidative stress might be a potentially useful strategy for the treatment of AD.
8-Hydroxy-2'-Deoxyguanosine↗
minK-related peptide 1 associates with Kv4.2 and modulates its gating function: potential role as beta subunit of cardiac transient outward channel?
Inherited mutations and a polymorphism in minK-related peptide 1 (MiRP1) have been linked to congenital or acquired long-QT syndrome, pointing to the importance of MiRP1 in maintaining the cardiac electrical stability. We tested whether MiRP1 could affect the function of Kv4.x (x=2 and 3), the major pore-forming (alpha) subunits of transient outward (I(to)) channels in the heart. We used the Xenopus oocyte expression system to examine the effects of MiRP1 on Kv4.x channel gating kinetics and current amplitude and correlated these effects with MiRP1 expression level. MiRP1 slowed the rates of Kv4.2 activation and inactivation and shifted the voltage dependence of channel gating in the positive direction. These effects had a similar "dose" dependence: they plateaued at a cRNA ratio (MiRP1:Kv4.2) of 13:1, with half-maximum effects at estimated cRNA ratios of 2 to 4. On the other hand, MiRP1 had no significant effects on Kv4.2 current amplitude in the same range of expression level. When expressed at a comparable low level, MiRP1 had similar (although smaller) effects on Kv4.3 but could not modulate Kv1.4 (another alpha subunit of I(to) channels in the heart). Kv4.2 could be coimmunoprecipitated with epitope-tagged MiRP1, indicating that the 2 could form a stable complex. Our data suggest that MiRP1 may serve as a regulatory (beta) subunit of I(to) channels in the heart. This is supported by the observation that MiRP1 induced an "overshoot" of Kv4.2 current amplitude during channel recovery from inactivation, similar to the overshoot of I(to) described for human epicardial myocytes.
4-Aminopyridine↗
Aldehydes potentiate alpha(2)(I) collagen gene activity by JNK in hepatic stellate cells.
Hepatic stellate cells (HSCs) are responsible for type I collagen deposition in liver fibrosis that leads to cirrhosis. The purpose of this study was to examine potential molecular signals that lead to increased alpha(2)(I) collagen gene expression by acetaldehyde, the primary metabolite of alcohol and malondialdehyde (MDA), a lipid peroxidation product known to be associated with chronic liver injury. MDA and the combination of MDA and acetaldehyde were employed to determine the effect on alpha(2)(I) collagen gene expression as assessed by transient transfection analysis and reverse transcriptase polymerase chain reaction (RT-PCR). Immunoblot and subsequent immunoprecipitation analysis examined stress-activated protein kinase (SAPK) activity. Cotransfection with a dominant negative mutant for c-jun nuclear kinase (dnJNK1) was also employed with the alpha(2)(I) collagen promoter. MDA increased alpha(2)(I) collagen gene expression nearly 2.5- to 3-fold, however there was no synergistic effect of the combination of acetaldehyde and MDA on alpha(2)(I) collagen gene activation and expression. Acetaldehyde, MDA, or both significantly increased JNK activity when compared to untreated stellate cells. The dnJNK1 expression vector abrogated alpha(2)(I) collagen transgene activity. In conclusion, JNK activation appears to be critical in the signaling cascade of oxidative metabolites of chronic alcohol-related liver injury and collagen gene activation.
Acetaldehyde↗
Selective ablation of retinoid X receptor alpha in hepatocytes impairs their lifespan and regenerative capacity.
Retinoid X receptors (RXRs) are involved in a number of signaling pathways as heterodimeric partners of numerous nuclear receptors. Hepatocytes express high levels of the RXRalpha isotype, as well as several of its putative heterodimeric partners. Germ-line disruption (knockout) of RXRalpha has been shown to be lethal in utero, thus precluding analysis of its function at later life stages. Hepatocyte-specific disruption of RXRalpha during liver organogenesis has recently revealed that the presence of hepatocytes is not mandatory for the mouse, at least under normal mouse facility conditions, even though a number of metabolic events are impaired [Wan, Y.-J., et al. (2000) Mol. Cell. Biol. 20, 4436-4444]. However, it is unknown whether RXRalpha plays a role in the control of hepatocyte proliferation and lifespan. Here, we report a detailed analysis of the liver of mice in which RXRalpha was selectively ablated in adult hepatocytes by using the tamoxifen-inducible chimeric Cre recombinase system. Our results show that the lifespan of adult hepatocytes lacking RXRalpha is shorter than that of their wild-type counterparts, whereas proliferative hepatocytes of regenerating liver exhibit an even shorter lifespan. These lifespan shortenings are accompanied by increased polyploidy and multinuclearity. We conclude that RXRalpha plays important cell-autonomous function(s) in the mechanism(s) involved in the lifespan of hepatocytes and liver regeneration.
Animals↗
Most central nervous system D2 dopamine receptors are coupled to their effectors by Go.
We reported previously that Go-deficient mice develop severe neurological defects that include hyperalgesia, a generalized tremor, lack of coordination, and a turning syndrome somewhat reminiscent of unilateral lesions of the dopaminergic nigro-striatal pathway. By using frozen coronal sections of serially sectioned brains of normal and Go-deficient mice, we studied the ability of several G protein coupled receptors to promote binding of GTPgammaS to G proteins and the ability of GTP to promote a shift in the affinity of D2 dopamine receptor for its physiologic agonist dopamine. We found a generalized, but not abolished reduction in agonist-stimulated binding of GTPgammaS to frozen brain sections, with no significant left-right differences. Unexpectedly, the ability of GTP to regulate the binding affinity of dopamine to D2 receptors (as seen in in situ [(35)S]sulpiride displacement curves) that was robust in control mice, was absent in Go-deficient mice. The data suggest that most of the effects of the Gi/Go-coupled D2 receptors in the central nervous system are mediated by Go instead of Gi1, Gi2, or Gi3. In agreement with this, the effect of GTP on dopamine binding to D2 receptors in double Gi1 plus Gi2- and Gi1 plus Gi3-deficient mice was essentially unaffected.
Animals↗
DNA synthesis and mitotic clonal expansion is not a required step for 3T3-L1 preadipocyte differentiation into adipocytes.
Upon differentiation induction of 3T3-L1 preadipocytes by a hormone mixture containing 1-isobutyl-3-methylxanthine, dexamethasone, and insulin, the preadipocytes undergo approximately 2 rounds of mitotic clonal expansion, which just precedes the adipogenic gene expression program and has been thought to be an essential early step for differentiation initiation. By inducing 3T3-L1 preadipocytes with each individual hormone, it was determined that the mitotic clonal expansion was induced only by insulin and not by 1-isobutyl-3-methylxanthine or dexamethasone. Cell number counting and fluorescence-activated cell-sorting analysis indicated that a significant fraction of 3T3-L1 preadipocytes differentiated into adipocytes without mitotic clonal expansion when induced with the combination of 1-isobutyl-3-methylxanthine and dexamethasone. Furthermore, when normally induced 3T3-L1 preadipocytes were treated with PD98059 (an inhibitor of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1) to block the activation of extracellular signal-regulated kinase (Erk) 1 and Erk2, the mitotic clonal expansion was blocked, but adipocyte differentiation was not affected. These observations were confirmed by bromodeoxyuridine labeling. The differentiated adipocytes induced with 1-isobutyl-3-methylxanthine and dexamethasone or standard hormone mixture plus PD98059 were not labeled by bromodeoxyuridine. Thus, it is evident that 3T3-L1 preadipocytes could differentiate into adipocytes without DNA synthesis and mitotic clonal expansion. Our results also suggested that activation of Erk1 and Erk2 is essential to but not sufficient for induction of mitotic clonal expansion.
1-Methyl-3-isobutylxanthine↗
Genome-wide analysis of developmental and sex-regulated gene expression profiles in Caenorhabditis elegans.
We have constructed DNA microarrays containing 17,871 genes, representing about 94% of the 18,967 genes currently annotated in the Caenorhabditis elegans genome. These DNA microarrays can be used as a tool to define a nearly complete molecular profile of gene expression levels associated with different developmental stages, growth conditions, or worm strains. Here, we used these full-genome DNA microarrays to show the relative levels of gene expression for nearly every gene during development, from eggs through adulthood. These expression data can help reveal when a gene may act during development. We also compared gene expression in males to that of hermaphrodites and found a total of 2,171 sex-regulated genes (P < 0.05). The sex-regulated genes provide a global view of the differences between the sexes at a molecular level and identify many genes likely to be involved in sex-specific differentiation and behavior.
Aging↗
Impaired adipogenesis and lipolysis in the mouse upon selective ablation of the retinoid X receptor alpha mediated by a tamoxifen-inducible chimeric Cre recombinase (Cre-ERT2) in adipocytes.
Retinoid X receptor alpha (RXRalpha) is involved in multiple signaling pathways, as a heterodimeric partner of several nuclear receptors. To investigate its function in energy homeostasis, we have selectively ablated the RXRalpha gene in adipocytes of 4-week-old transgenic mice by using the tamoxifen-inducible Cre-ERT2 recombination system. Mice lacking RXRalpha in adipocytes were resistant to dietary and chemically induced obesity and impaired in fasting-induced lipolysis. Our results also indicate that RXRalpha is involved in adipocyte differentiation. Thus, our data demonstrate the feasibility of adipocyte-selective temporally controlled gene engineering and reveal a central role of RXRalpha in adipogenesis, probably as a heterodimeric partner for peroxisome proliferator-activated receptor gamma.
Adipocytes↗
Formation of novel polymeric nanoparticles.
We have found that not only block copolymers but also ionomers can self-assemble in a selective solvent to form surfactant-free nanoparticles. The self-assembly can be induced by chemical reaction, polymer-polymer complexation, and microphase inversion in addition to the temperature. A recently developed microwave method for the preparation of uniform surfactant-free polymeric nanoparticles is also reviewed. Our results have revealed that for a given dispersion, the particle surface area occupied per stabilizer (surfactant, polymer chains, and ionic groups) is close to a constant.
Microspheres↗