Biomedical subjects
Publications and source records attributed to Y Xia.
In our search for novel anti-HIV agents, seven 17-carboxylated steroid derivatives were synthesized and evaluated as potential anti-HIV agents. Compound 13 exhibited potent anti-HIV activity in acutely infected H9 lymphocytes with EC50 and therapeutic index values of 0.8 microM and 300, respectively.
Intracellular recording methods and immunostaining revealed the existence of functional group I metabotropic glutamate receptors (mGluRs) in submucous plexus neurons of guinea-pig ileum. Selective group I, but not groups II or III metabotropic glutamate receptor agonists induced concentration-dependent, slowly-activating depolarizing responses. Group I metabotropic glutamate receptor antagonism observed with (S)-4-carboxyphenylglycine (S-4-CPG) (100 - 600 microM) was competitive as determined by Schild analysis (pA(2)=3.81+/-0.02). Neither the group II and III metabotropic nor ionotropic glutamate receptor antagonists altered responses evoked by group I receptor agonists. Immunoreactivities for metabotropic glutamate 1alpha and 5 receptors were found to locate exclusively in neurons in the submucous plexus of guinea-pig ileum with the highest density around the cell bodies. The results suggest that group I metabotropic glutamate receptors are functionally expressed in the submucous plexus of guinea-pig small intestine.
Thrombopoietin (TPO) is the primary regulator of platelet production and acts through binding its receptor, c-mpl, found on megakaryocyte progenitor cells, megakaryocytes and platelets. Circulating levels of TPO are regulated primarily by the clearance of TPO after it binds to c-mpl receptors on circulating platelets. In this study the interaction of TPO with the platelet c-mpl receptor has been analysed under physiological conditions using radiochemical and pharmacokinetic approaches. 125I-rHuTPO was prepared using a novel method of gentle iodination that preserved its biological activity and used to demonstrate that platelets, but not endothelial cells, have a single class of binding sites (56 +/- 17 binding sites/platelet) with high affinity (Kd = 163 +/- 31 pM). Cross-linking experiments confirmed that TPO, but not erythropoietin (EPO), specifically associated with the 95 kD platelet c-mpl receptor. Upon addition of TPO to platelets, 80% of the TPO binding sites were internalized within an hour and were not recycled. TPO that was not bound by platelets was stable for up to 6 d in both platelet-poor and platelet-rich plasma. Using unlabelled recombinant human TPO (rHuTPO), standard pharmacokinetic analysis demonstrated that platelets have an average TPO clearance of 1.24 +/- 0.38 ml/h/109 platelets and that TPO clearance was reduced by low temperature but not by a number of drugs or metabolic inhibitors. The maximal amount of TPO removed by platelets in vitro was identical to that predicted by the total number of TPO binding sites. These results provide a biochemical and pharmacokinetic basis for the clinical use of TPO and for understanding possible disorders of platelet production.
BACKGROUND: Fractalkine is a newly identified T-cell and monocyte/macrophage (Mphi) chemokine with a transmembrane domain and is a cell-surface protein on activated endothelium. It can mediate adhesion of cells expressing the fractalkine receptor CX3CR1. These unique features make fractalkine well suited for leukocyte recruitment in tissues with high blood flow as in the renal glomerulus. METHODS: Fractalkine expression in glomeruli and response of isolated glomerular inflammatory cells to fractalkine were studied in the Wistar-Kyoto (WKY) crescentic glomerulonephritis model. Antibody was used to confirm the proinflammatory role of fractalkine. RESULTS: Fractalkine was markedly induced in the endothelium of nephritic rat glomeruli, and inflammatory leukocytes infiltrating the glomeruli expressed increased levels of CX3CR1. Anti-CX3CR1 antibody treatment dramatically blocked leukocyte infiltration in the glomeruli, prevented crescent formation, and improved renal function. CONCLUSIONS: Fractalkine plays a central role in leukocyte trafficking at the endothelium in the high-flow glomerular circuit and, in turn, implicates CX3CR1 as a prime drug target for therapeutic intervention of endothelium-related inflammatory diseases.
OBJECTIVE: This study was undertaken to investigate the metabolism of selenite in men with life-long intakes of deficient, adequate and excess selenium. METHODS: Stable isotopes of selenium were infused for five hours into Chinese men living in deficient, adequate or excessive selenium areas, and 24-hour urine and blood samples were collected daily for the next seven days. Stable isotopic selenium excretion was determined in urine and in whole plasma and plasma fractions. RESULTS: Even though there was a positive correlation of selenium intake with the urinary excretion of this element, this relationship was not linear over the entire range (deficient, adequate, excessive) of selenium intake. When the urine excretion was normalized internally within each group, a sharp increase in the slope of this relationship was found when long-term intake increased to adequate amounts, but the slope reached a plateau when the daily intake exceeded the adequate group. The plasma selenoprotein P fraction was labeled initially, but the incorporation in the glutathione peroxidase fraction subsequently increased by a small amount. A two-month dietary restriction of selenium of the subjects from the excess area did not result in a reduction of urinary excretion of infused selenite. CONCLUSION: A complex relationship exists between long-term intake of selenium and selenium status, and subjects living in the excess area are more saturated with selenium than anticipated. More than two months of depletion are required to affect urinary excretion of selenium.
Children putatively immune to the large roundworm Ascaris lumbricoides were identified in an area of Nigeria where infection is hyperendemic. Immunity was associated with higher levels of serum ferritin, C-reactive protein, and eosinophil cationic protein, indicating ongoing acute phase or inflammatory processes. In contrast, children who were susceptible to the infection had little serological evidence of inflammation despite their high parasite burdens. Immunoglobulin G (IgG) antibody activity in all subclasses was present in high titer in most children but appeared to have no protective function. Despite exceptionally high total IgE levels, there was no evidence that atopic responses to local common allergens was associated with natural immunity to Ascaris. Among those individuals who produced IgG antibody to recombinant ABA-1 allergen of Ascaris, the naturally immune group had significantly more IgE antibody to the allergen than did those susceptible to the infection. IgE antibody responses in conjunction with innate inflammatory processes therefore appear to associate with natural immunity to ascariasis.
Members of the NF-kappaB/RelB family of transcription factors play important roles in the regulation of inflammatory and immune responses. RelB, a member of this family, has been characterized as a transcription activator and is involved in the constitutive NF-kappaB activity in lymphoid tissues. However, in a previous study we observed an overexpression of chemokines in RelB-deficient fibroblasts. Here we show that RelB is an important transcription suppressor in fibroblasts which limits the expression of proinflammatory mediators and may exert its function by modulating the stability of IkappaBalpha protein. Fibroblasts from relb(-/-) mice overexpress interleukin-1alpha (IL-1alpha), IL-1beta, and tumor necrosis factor alpha in response to lipopolysaccharide (LPS) stimulation. These cells have an augmented and prolonged LPS-inducible IKK activity and an accelerated degradation which results in a diminished level of IkappaBalpha protein, despite an upregulated IkappaBalpha mRNA expression. Consequently, NF-kappaB activity was augmented and postinduction repression of NF-kappaB activity was impaired in these cells. The increased kappaB-binding activity and cytokine overexpression was suppressed by introducing RelB cDNA or a dominant negative IkappaBalpha into relb(-/-) fibroblasts. Our findings suggest a novel transcription suppression function of RelB in fibroblasts.
An approach to construct low resolution models of protein structure from sequence information using a combination of different methodologies is described. All possible compact self-avoiding C alpha conformations (approximately 10 million) of a small protein chain were exhaustively enumerated on a tetrahedral lattice. The best scoring 10,000 conformations were selected using a lattice-based scoring function. All-atom structures were then generated by fitting an off-lattice four-state phi/psi model to the lattice conformations, using idealised helix and sheet values based on predicted secondary structure. The all-atom conformations were minimised using ENCAD and scored using a second hybrid scoring function. The best scoring 50, 100, and 500 conformations were input to a consensus-based distance geometry routine that used constraints from each the conformation sets and produced a single structure for each set (total of three). Secondary structures were again fitted to the three structures, and the resulting structures were minimised and scored. The lowest scoring conformation was taken to be the "correct" answer. The results of application of this method to twelve proteins are presented.
Conventional intracellular microelectrodes and injection of biocytin were used to study the actions of IL-1beta and IL-6 on electrical and synaptic behavior in morphologically identified guinea pig small intestinal submucous neurons. Exposure to nanomolar concentrations of either IL-1beta or IL-6 stimulated neuronal excitability. The excitatory action consisted of depolarization of the membrane potential, decreased membrane conductance, and increased discharge of action potentials. Excitatory action of IL-1beta was suppressed by the natural IL-1beta human receptor antagonist. Electrical stimulation of sympathetic postganglionic axons evoked inhibitory postsynaptic potentials (IPSPs), and stimulation of cholinergic axons evoked nicotinic fast excitatory postsynaptic potentials (EPSPs). Both kinds of synaptic potentials occurred in neurons with uniaxonal morphology believed to be secretomotor neurons. Either IL-1beta or IL-6 suppressed the noradrenergic IPSPs and the fast EPSPs, and the two acted synergistically when applied in combination. Suppression of the IPSP resulted from presynaptic inhibition of the release of norepinephrine from sympathetic nerves. The results suggest that the presence of either or both inflammatory cytokines will release the sympathetic brake from secretomotor neurons to the intestinal crypts and from nicotinic synapses in the integrative microcircuits, where norepinephrine is known to have a presynaptic inhibitory action. This, in concert with excitation of secretomotor neurons, may lead to neurogenic secretory diarrhea.
Compounds that interact with DNA or microtubules by multiple mechanisms and cause diverse cytotoxic lesions are potential targets for anticancer drug development. Accordingly, a relatively new approach to the rational design of antitumor agents is based on the quinolone class of antibacterials. Their mechanism of antibacterial action involves inhibition of DNA gyrase, and numerous new quinolones do exhibit antitumor activity. Thus, these new quinolone structures display a novel mode of action for the quinolone class as antitumor agents. The potential for quinolones to be used as topoisomerase II inhibitors, as well as antimitotic agents, is reviewed with a focus on recent discoveries and development of antitumor quinolones, especially related work in the author's laboratory.
OBJECTIVE: To investigate the prognostic significance of DNA image cytophotometric data. STUDY DESIGN: Twenty-six osteosarcomas in patients without lung metastases were investigated for several cytophotometric data. In 24 cases, these data were correlated with the clinical course of the patients to assess the prognostic value of nuclear DNA content in osteosarcomas. RESULTS: Of all osteosarcomas, 96% showed aneuploid DNA content. Patients with tumors having a 2c deviation index (2cDI) of 12.00, DNA malignancy grade (DNA-MG) of 2.0, a mean DNA content (MDC) of 4.95 c, DNA index (DI) of 1.75 or mean nuclear area (MNA) of 130 microns 2 had a significantly lower overall survival rate as compared to those with lower values (P < .05). CONCLUSION: Image cytophotometric features, such as 2cDI, DNA-MG, MDC, DI and MNA, are of prognostic value in patients with osteosarcoma and free of lung metastases.
The purpose of the present study was to investigate the effects of Se restriction on the excretion of Se in men who had consumed high levels of this element during their entire lives. With the use of stable isotopes of Se as selenite, the excretion of methylated Se in urine was investigated in Chinese men (n 10) who had habitual chronic high intakes of this element. The relationship between either urine Se or trimethylselenonium (TMSe) to the estimated long-term Se intake was not linear over the entire range of intake, which was also true for the infusion of labelled selenite. A non-linear relationship was also found between urine TMSe and urine Se both for TMSe arising from catabolism of endogenous body Se and that from infused selenite. The data suggest a close precursor-product relationship of urine Se and its TMSe component based on the nearly identical specific activities for these two selenocompounds. Although dimethylselenide in breath was not measured in the present study, combining urinary TMSe with this breath test may be more useful in the assessment of long-term Se status.
The structure of a synthesized peptide with the sequence of NHILPNESAYDQKNIRRRVYDALNVLMAMNIISK that corresponds to residues 151-184 of transcription factor DP-1 (Girling et al., Nature 362 (1993) 83-87) was determined by 1H-nuclear magnetic resonance in water and 40% d3-trifluoroethanol/water, respectively. Nuclear Overhauser effect cross peaks, alphaH chemical shifts and J-coupling constants of alphaH-NH show that the peptide consists a helix from Ser-8 to Ser-33 in solution. Fifty structures were constructed with 288 upper distance limits and 21 angle constraints by DIANA (Guntert et al., J. Mol. Biol. 217 (1991) 517-530). Although the N-terminal of the peptide exhibits a random conformation, the 20 best structures show a root mean square deviation of 0.89+/-0.36 A for backbone atoms and 1.80+/-0.34 A for heavy atoms from residue Ser-8 to Ser-33. This result supports the proposal that DP-1 and E2F-1 may dimerize with a coiled-coil type interaction.
MAP kinase (MAPK) cascades are composed of a MAPK, MAPK kinase (MAPKK), and a MAPKK kinase (MAPKKK). Despite the existence of numerous components and ample opportunities for crosstalk, most MAPKs are specifically and distinctly activated. We investigated the basis for specific activation of the JNK subgroup of MAPKs. The specificity of JNK activation is determined by the MAPKK JNKK1, which interacts with the MAPKKK MEKK1 and JNK through its amino-terminal extension. Inactive JNKK1 mutants can disrupt JNK activation by MEKK1 or tumor necrosis factor (TNF) in intact cells only if they contain an intact amino-terminal extension. Mutations in this region interfere with the ability of JNKK1 to respond to TNF but do not affect its activation by physical stressors. As JNK and MEKK1 compete for binding to JNKK1 and activation of JNKK1 prevents its binding to MEKK1, activation of this module is likely to occur through sequential MEKK1:JNKK1 and JNKK1:JNK interactions. These results underscore a role for the amino-terminal extension of MAPKKs in determination of response specificity.
The structure of a synthesized peptide with the sequence GVVDLNWAAEVLKVQKRRIYDITNVLEGIQ which corresponds to residues 149-178 of transcription factor E2F-1 was determined by 1H nuclear magnetic resonance in 40% d3-TFE/water. NOE cross peaks and alphaH chemical shifts indicate that the peptide consists of a helix from Ala-8 to Leu-26 in this solution. Circular dichroism measurements confirm the presence of nearly 45% helix in TFE/water solution but show no evidence of helicity in water solution of this peptide. Fifty structures were constructed with 329 upper distance limits by DIANA. The 20 best conformers show a RMSD of 0.78 A for backbone atoms and 1.78 A for heavy atoms from residue Ala-8 to Leu-26. This result, together with our previous work on the solution structure of a fragment of DP-1, supports the proposal that E2F-1 and DP-1 may dimerize with a coiled-coil type interaction.
It has been previously shown that besides synthesizing nitric oxide (NO), neuronal and inducible NO synthase (NOS) generates superoxide (O-2) under conditions of L-arginine depletion. However, there is controversy regarding whether endothelial NOS (eNOS) can also produce O-2. Moreover, the mechanism and control of this process are not fully understood. Therefore, we performed electron paramagnetic resonance spin-trapping experiments to directly measure and characterize the O-2 generation from purified eNOS. With the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), prominent signals of O-2 adduct, DMPO-OOH, were detected from eNOS in the absence of added tetrahydrobiopterin (BH4), and these were quenched by superoxide dismutase. This O-2 formation required Ca2+/calmodulin and was blocked by the specific NOS inhibitor N-nitro-L-arginine methyl ester (L-NAME) but not its non-inhibitory enantiomer D-NAME. A parallel process of Ca2+/calmodulin-dependent NADPH oxidation was observed which was also inhibited by L-NAME but not D-NAME. Pretreatment of the enzyme with the heme blockers cyanide or imidazole also prevented O-2 generation. BH4 exerted dose-dependent inhibition of the O-2 signals generated by eNOS. Conversely, in the absence of BH4 L-arginine did not decrease this O-2 generation. Thus, eNOS can also catalyze O-2 formation, and this appears to occur primarily at the heme center of its oxygenase domain. O-2 synthesis from eNOS requires Ca2+/calmodulin and is primarily regulated by BH4 rather than L-arginine.
The members of deiodinase family including type I, type II and type III deiodinase are seleno-proteins. They play an important role in the regulation of thyroid hormone metabolism. They are different in substrate specificity and sensitivity to inhibitor and kinetics mechanism. Their cDNAs exhibit limited regions of homology, but they have common conserved sequence, TGA codon of selenocysteine and some structures and sequences which are essential for selenocysteine insertion.
Postischemic endothelial dysfunction may occur as a result of the effects of endogenous oxidants like hydrogen peroxide. Since endothelium-dependent vasodilator function may be affected by pHi, the effect of hydrogen peroxide on endothelial pHi was examined. Hydrogen peroxide (100 micromol/L for 10 minutes) decreased pHi from 7.24+/-0.01 to 7.02+/-0.02 and inhibited recovery from an ammonium chloride-induced intracellular acid load in carboxy SNARF 1 (c-SNARF 1)-loaded human aortic endothelial cells in bicarbonate-free solution. Prior inhibition of Na+/H+ exchange with 5-(N-ethyl-N-isopropyl)amiloride (10 micromol/L), by removal of extracellular Na+, or by glycolytic inhibition with iodoacetic acid blocked the subsequent effect of hydrogen peroxide on pHi. A 2-minute exposure to 100 micromol/L H2O2 decreased intracellular ATP levels by approximately 40%; this was prevented by 3-aminobenzamide and nicotinamide (1 mmol/L each), inhibitors of the DNA repair enzyme poly(ADP-ribose) polymerase. Both 3-aminobenzamide and nicotinamide significantly inhibited the hydrogen peroxide-induced intracellular acidification and the effect of hydrogen peroxide on recovery from an intracellular acid load. Hydrogen peroxide decreases pHi in human endothelial cells by inhibiting Na+/H+ exchange. This appears to be mediated by activation of the DNA repair enzyme poly(ADP-ribose) polymerase and subsequent depletion of intracellular ATP. Since a decrease in pHi in this range may alter the activity of NO synthase or affect the synthesis of vasodilator prostaglandins, the effect of hydrogen peroxide on the endothelial Na+/H+ exchanger may be important in the pathogenesis of postischemic endothelial dysfunction.