Biomedical subjects
Publications and source records attributed to P Li.
We investigated sex- or gender-dependent differences in nociception using the formalin test in mice. In addition to typical biphasic responses, a new, late phase (Phase 3) was observed. A local anesthetic QX-314 injected at the end of Phase 2 blocked Phase 3. Phase 3 in female mice was significantly greater than that in male mice regardless the stage of the estrus cycle.
Nociceptin (orphanin FQ), the newly discovered endogenous ligand for the novel opioid receptor-like 1 receptor, has been initially found to participate in pain modulation. In this study, centrally mediated cardiovascular actions of this peptide were investigated in the alpha-chloralose/urethane-anesthetized rats. We found that bilateral injection of nociceptin (10 nmol) into the rostral ventrolateral medulla (RVLM), wherein injection of excitatory amino acid dl-homocysteic acid (3 nmol) induced typical pressor responses, significantly reduced arterial blood pressure and heart rate by -32% and -15%, respectively. Reduction of blood pressure and heart rate in response to intra-RVLM injection of nociceptin was dose-dependent with a threshold dose being 3 nmol. Pretreatment with the selective nociceptin receptor antagonist, [Phe1psi(CH2-NH)Gly2]NC(1-13)NH2 (10 nmol), into the RVLM abolished the nociceptin-induced cardiovascular inhibition. In contrast, non-selective opioid receptor antagonist, naloxone (10 nmol), did not modify the hypotension and bradycardia induced by nociceptin, even though naloxone at the same dose prevented reduction of blood pressure and heart rate induced by intra-RVLM injection of methionine-enkephalin (3 nmol). Both [Phe1psi(CH2-NH)Gly2]NC(1-13)NH2 and naloxone injection alone had no significant effect on baseline blood pressure and heart rate. These data suggest that the newly discovered opioid-like neuropeptide nociceptin in the CNS exert powerful influence on hemodynamic activity by affecting the RVLM neurons. This influence is inhibitory in nature, which may not be active in normal physiological conditions. Moreover, the cardiovascular effects of nociceptin were mediated by activation of specific nociceptin receptors rather than typical naloxone-sensitive opioid receptors.
Coexpression of different effector molecules from a single vector (a dual-function vector) may provide enhanced efficacy. Thus far most of the reported anti-HIV dual-function vectors express different effector RNAs as a chimeric molecule. In our study involving retroviral vectors coexpressing a U5 ribozyme and either an anti-tat or anti-rev antisense RNA, chimeric vectors exhibit poor potency in several important functional aspects, including inhibition of HIV replication, protection against cytopathic effects, and suppression of target gene function. Surprisingly, such a poor efficacy of chimeric vector function was not associated with a lower level of effector RNA expression. These results indicate that expression of two effector RNAs as a chimeric molecule can lead to interference, reducing their global biological effects. More importantly, we have demonstrated that such interference can be avoided by coexpressing these effector RNAs as separate molecules through a new dual-function vector, called a dual-effector cassette (Dec) vector, developed in this study. We also define some of the design alterations that might affect the efficacy of the Dec vector and demonstrate that forward-designed Dec vectors are more efficacious than reverse-designed Dec vectors, which express a lower level of effector RNA owing to the instability of the 5' effector cassettes in the provirus. We believe that the principle of Dec vector design may also be applicable for the coexpression of other therapeutic RNA effectors in many gene therapy applications.
Since starch synthases IIa (SSIIa) and SSIIb have not been purified from plant tissue, their structure-function relationships have not been well characterized. Therefore, we have expressed these SS genes in Escherichia coli, purified them to apparent homogeneity, and studied their kinetic properties. In addition, the N-terminally truncated forms of these enzymes were studied in an attempt to understand the function of the diverse N-terminal sequences in SS. Our results show that, like SSI, the N-terminal extensions of SSIIa and SSIIb are not essential for catalytic activity and no extensive changes in their kinetic properties are observed upon their N-terminal truncation. Each isoform of SS can be distinguished based on its kinetic properties. Maize SSI and maize SSIIb exhibit higher Vmax with glycogen as a primer, while the converse is true for SSIIa. However, the specific activity of SSIIb is at least two- to threefold higher than that for either SSI or SSIIa. Although SSIIb exhibits the highest maximal velocity of the isoforms compared, its apparent affinity for primer is twofold lower than the affinity of SSI and SSIIa for primer. Perhaps these differences in primer affinity, primer preference, and maximal velocities all contribute in some way to the different structure(s) of starch during its synthesis. Expression and purification of maize SS has now provided us a useful tool to address the role(s) of SS in starch synthesis and starch structure.
The separation of macromolecules such as polymers and DNA by means of electrophoresis, gel permeation chromatography or filtration exploits size-dependent differences in the time it takes for the molecules to migrate through a random porous network. Transport through the gel matrices, which usually consist of full swollen crosslinked polymers, depends on the relative size of the macromolecule compared with the pore radius. Sufficiently small molecules are thought to adopt an approximately spherical conformation when diffusing through the gel matrix, whereas larger ones are forced to migrate in a snake-like fashion. Molecules of intermediate size, however, can get temporarily trapped in the largest pores of the matrix, where the molecule can extend and thus maximize its conformational entropy. This 'entropic trapping' is thought to increase the dependence of diffusion rate on molecular size. Here we report the direct experimental verification of this phenomenon. Bragg diffraction from a hydrogel containing a periodic array of monodisperse water voids confirms that polymers of different weights partition between the hydrogel matrix and the water voids according to the predictions of the entropic trapping theory. Our approach might also lead to the design of improved separation media based on entropic trapping.
Glutamate, the major excitatory neurotransmitter in the central nervous system, activates three different receptors that directly gate ion channels, namely receptors for AMPA (alpha-amino-3-hydroxy-5-methyl isoxozole propionic acid), NMDA (N-methyl-D-aspartate), and kainate, a structural analogue of glutamate. The contribution of AMPA and NMDA receptors to synaptic transmission and plasticity is well established. Recent work on the physiological function of kainate receptors has focused on the hippocampus, where repetitive activation of the mossy-fibre pathway generates a slow, kainate-receptor-mediated excitatory postsynaptic current (EPSC). Here we show that high-intensity single-shock stimulation (of duration 200 microseconds) of primary afferent sensory fibres produces a fast, kainate-receptor-mediated EPSC in the superficial dorsal horn of the spinal cord. Activation of low-threshold afferent fibres generates typical AMPA-receptor-mediated EPSCs only, indicating that kainate receptors may be restricted to synapses formed by high-threshold nociceptive (pain-sensing) and thermoreceptive primary afferent fibres. Consistent with this possibility, kainate-receptor-mediated EPSCs are blocked by the analgesic mu-opiate-receptor agonist Damgo and spinal blockade of both kainate and AMPA receptors produces antinociception. Thus, spinal kainate receptors contribute to transmission of somatosensory inputs from the periphery to the brain.
The recently available antagonist selective for novel nociceptin receptor, [Phe1 psi(CH2-NH)Gly2]NC(1-13)NH2, was utilized in this study to verify specificity of nociceptin receptor in mediating the nociceptin-induced inhibition of electrical activity of neurons in the rostral ventrolateral medulla of rat brain slices. Perfusion of nociceptin (10 nM) considerably reduced spontaneously firing frequency of the medullary neurons. Co-perfusion of [Phe1 psi(CH2-NH)Gly2]NC(1-13)NH2 (10 microM) completely blocked the nociceptin-induced depression of the neuronal activity. Blocking effect of [Phe1 psi(CH2-NH)Gly2]NC(1-13)NH2 was concentration-dependent. However, the nociceptin antagonist did not modify basal, and opioid peptide enkephalin-depressed, firing rates of the neurons. In contrast to [Phe1 psi(CH2-NH)Gly2]NC(1-13)NH2, the non-selective opioid receptor antagonist naloxone (10 microM) failed to affect the nociceptin inhibition even though naloxone at a lower concentration (1 microM) readily blocked enkephalin-induced depression of the neuronal activity. These data indicate that the nociceptin-induced inhibition of spontaneous discharge of the rostral ventrolateral medulla neurons is specifically mediated by [Phe1 psi(CH2-NH)Gly2]NC(1-13)NH2-sensitive nociceptin receptors distinct from typical naloxone-sensitive opioid receptors.
The role of the CD45-associated phosphoprotein (LPAP / CD45-AP) during an immune response remains unclear. To understand better the function of LPAP we generated LPAP-deficient mice by disrupting exon 2 of the LPAP gene. LPAP-null mice were healthy and did not show gross abnormalities compared to their wild-type littermates. However, immunofluorescence analysis of T and B lymphocytes revealed a reduced expression of CD45, which did not affect a particular subpopulation. In contrast to a recent report (Matsuda et al., J. Exp. Med. 1998. 187: 1863 - 1870) we neither observed significant alterations of the assembly of the CD45 / lck-complex nor of polyclonal T-cell responses. However, lymphnodes from LPAP-null mice showed increased cellularity, which could indicate that expression of LPAP might be required to prevent expansion of lymphocytes in particular lymphatic organs rather than potentiating immune responses.
OBJECTIVE: Inappropriate telomerase expression has been reported to be associated with the development and/or progression of malignancies. Therefore, the purpose of the study was to determine and evaluate the levels of telomerase activity in normal ovary, cystadenomas, low-malignant-potential tumors, and carcinomas of the ovary. METHODS: In the present study, telomerase activity was examined in frozen tissue specimens of normal ovary (n = 6), ovarian cystadenomas (n = 13), ovarian low-malignant-potential (LMP) tumors (n = 12), and ovarian invasive carcinomas (n = 81). Clinicopathological information including age at diagnosis, histological grade, FIGO stage, presence of distant metastasis at diagnosis, and residual disease was available for all patients with ovarian carcinomas (n = 81). Telomerase activity was assessed by the telomeric repeat amplification protocol (TRAP). Arbitrary values were assigned to processivity of telomerase activities based on the molecular weights of the telomeric repeat DNA ladders, and were graded as "negative," "moderate" (< or =99 bp), or "high" (>100 bp) activities. The specificity of telomerase activity was determined by the pretreatment of telomerase-positive control or tumor samples with RNase that led to the abolition of the activity. In addition, to determine the possibility of false negativity due to the presence of telomerase inhibitors, TRAP assay was performed on each of the telomerase-negative specimens by mixing them individually with the telomerase-positive control. RESULTS: Telomerase activity in the progression of ovarian carcinogenesis was evaluated. In comparison with normal ovary/cystadenoma (32%), a much higher frequency of the moderate activity was observed in LMP tumors (67%) or invasive carcinomas (57%), suggesting a close association between the latter two categories. The results reflect a subpopulation of telomerase-positive LMP tumor cells with the potential to develop invasive carcinomas. None of the specimens of the benign or LMP tumors exhibited high activity. In contrast, 18% of ovarian invasive carcinomas showed high telomerase activity (P = 0.013, Fisher exact test) and further 57%, moderate activity (75% in all). A statistically significant difference was observed in the expression of telomerase activity between normal ovary/benign cystadenomas and ovarian invasive carcinomas (P = 0.001, chi(2) test). CONCLUSIONS: The study showed a high prevalence of telomerase activity in LMP tumors or invasive carcinomas, the high levels of telomerase activity being associated exclusively with the invasive ovarian carcinomas. Therefore, the levels of processivity of telomerase activity and evidence of its statistically significant association with ovarian carcinoma suggest its role in the progression of ovarian carcinogenesis.
The sequence of a 900-nucleotide segment (encoding part of the reverse transcriptase, including the entire RNase H domain) of the pol gene of the murine leukaemia virus (MLV) amphotropic strain 4070A is presented. Alignment of the inferred 4070A RNase H amino acid sequence (157 residues) with other MLV RNase H sequences revealed only minor differences compared with the divergence between other retroviral and prokaryotic or eukaryotic RNase H sequences. Only 10 residues were invariant across the entire sample set, but secondary structure predictions for the enzymes from E. coli, yeast, human liver and diverse retroviruses (HIV, Rous sarcoma virus, foamy viruses) supported, in every case, the five beta-strands (1 to 5) and four or five alpha-helices (A, B/C, D, E) that have been identified by crystallography in the RNase H domain of HIV-1 reverse transcriptase and in E. coli RNase H. In the case of MLV, analysis of the RNase H domain sequences inferred from 10 different strains (including the amphotropic 4070A) predicted all five alpha-helices (A-E), as well as beta-strands 4 and 5. However, the N-terminal segment (residues 1-40) was predicted, without exception and with high probability, to fold uniquely into one (or two adjacent) alpha-helix(es) encompassing residues 13-37, instead of the three beta-strands known to exist in the HIV-1 and E. coli enzymes. The unerring consistency between the known and predicted structures of the HIV-1 and E. coli enzymes, and the prediction of the same structural elements (including beta-strands 1-3 within the N-terminal segment) for all other (non-MLV) RNase H proteins examined in this study, suggests that the N-terminal segment of the MLV RNase H domain assumes a conformation distinct from that of other retroviral and cellular RNase H molecules. An additional (sixth) beta-strand was also predicted uniquely within the C-terminal region of foamy virus RNase H domains.
BACKGROUND: To provide clinical research experience in community hospital programs, we integrated research training with the daily work of surgical residency. METHODS: Teaching conferences and daily care of patients served as a source of projects. Emphasizing team work, high-yield projects are assigned to principal resident investigators, who lead a project team. Regular meetings with the faculty provides guidance and supervision, working to meet an abstract deadline. RESULTS: Over a 3-year period, 10 of 16 trainees participated in at least one project. Thirteen projects were presented at national meetings and 13 articles (including 6 from presentations) were published. Three projects were abandoned for lack of progress, and 5 manuscripts have been rejected. CONCLUSION: The integrated program trains multiple residents/projects with low requirements in space and funding, but high demands of organization and faculty time.
BACKGROUND: Despite many randomized controlled trials, the merits of laparoscopic appendectomy remain unclear. A meta-analysis may provide insights not evident from any individual studies. DATA SOURCES: Systematic literature search yielded 17 trials (1,962 subjects) of true randomized design with usable statistical data comparing laparoscopic and conventional appendectomy in adults. The effect sizes for operating time, hospitalization, postoperative pain, return to normal activity, wound infection, and intra-abdominal abscess were calculated, using the random effects model to allow for heterogeneity. An estimate of the robustness of all positive findings was also calculated. RESULTS: Modest but statistically significant effect sizes were found for four of the six outcome measures. Laparoscopic appendectomy takes 31% longer to perform, but results in less postoperative pain, faster recovery (by 35%), and lower wound infection rates (by 60%). CONCLUSION: Laparoscopic appendectomy offers significant improvement in postoperative outcomes at the cost of a longer operation.
Binding of the Fc portion of IgG coated on targets to Fcgamma receptors (e.g., CD16) expressed on leukocytes (i.e., 2D binding) is an initiating step for immune responses such as phagocytosis or antibody-dependent cellular cytotoxicity. In vivo, circulating leukocytes are exposed to plasma IgG. The competition from soluble IgG (i.e., 3D binding) may affect the 2D binding. Many cell surface receptors, CD16 included, have soluble counterparts. While their physiological significance is not clear, receptor-based competitive inhibition therapy, in which soluble receptors, ligands, and their analogs are employed to compete with surface-bound receptors and ligands to prevent unwanted adhesion, is widely used to treat various diseases. To provide a quantitative basis for design of these therapeutic approaches, we developed a mathematical model for 2D and 3D competition binding. The model relates cell-surface adhesion (in the presence and absence of dislodging forces) to the concentration of the soluble competitor, the densities of the surface-bound receptors and ligands, as well as the binding affinities of the 2D and 3D interactions. Binding of CD16-expressing cells to an IgG-coated surface in the presence of a soluble competitor (IgG or anti-CD16 antibody) was quantified by a centrifugation assay. The agreement between experiment and theory supports the validity of the model, which could be useful in predicting the efficacy of the competitor.
The antithrombotic activity of low-molecular-weight heparin calcium (LMWH-Ca) was studied in venous and arterial thrombosis models, arterial thrombosis model in rats, arterio-venous shunt model and venous thrombosis model in rabbits. The data showed that LMWH-Ca reduced thrombus formation in a dose-dependent manner. It suggests that LMWH-Ca is a potent antithrombotic agent for venous thrombosis, and also may be a beneficial therapeutic agent in arterial thrombosis.
The classic opioid peptide, enkephalin, and the novel member of the opioid family, nociceptin/orphanin FQ, inhibit the spontaneous electrical activity of neurons recorded from the rostral ventrolateral medulla, presumably cardiovascular neurons. In this study, the putative effects of endomorphin-1 and endomorphin-2, the newly discovered endogenous ligands for the micro-opioid receptor, on the electrical activity of rostral ventrolateral medulla neurons were investigated in rat brain slices in vitro. Like enkephalin and nociceptin, perfusion of endomorphin-1 or endomorphin-2 profoundly inhibited spontaneous discharges of 43% and 38% of the medullary neurons, respectively. No excitatory response to perfusion of either endomorphin was found in all neurons surveyed. Both endomorphins produced concentration-dependent inhibition. However, endomorphin-1 was more potent than endomorphin-2 for production of the inhibition, as demonstrated by the greater and longer suppression induced by endomorphin-1 than that induced by endomorphin-2 at the same concentration. Among the four opioid agonists tested, EC50 values (in nM) were 3.17 (endomorphin-1), 3.02 (nociceptin), 10.1 (endomorphin-2) and 150.0 (enkephalin). The non-selective opioid receptor antagonist, naloxone, blocked the inhibitory responses of the neurons to endomorphin-1, endomorphin-2 and enkephalin, but not to nociceptin. The selective mu antagonist, beta-funaltrexamine, prevented the neuronal inhibition induced by endomorphins, but not by enkephalin and nociceptin. Neither naloxone nor beta-funaltrexamine alone had a significant effect on the firing rate of the neurons. These results demonstrate that endomorphin-1 and, to a lesser extent, endomorphin-2 exert an inhibitory modulation of the electrical activity of rostral ventrolateral medulla neurons, which is mediated through the stimulation of mu-opioid receptors.
The major histocompatibility complex (MHC) class I homolog MIC-A functions as a stress-inducible antigen that is recognized by a subset of gammadelta T cells independent of beta2-microglobulin and bound peptides. Its crystal structure reveals a dramatically altered MHC class I fold, both in detail and overall domain organization. The only remnant of a peptide-binding groove is a small cavity formed as the result of disordering a large section of one of the groove-defining helices. Loss of beta2-microglobulin binding is due to a restructuring of the interaction interfaces. Structural mapping of sequence variation suggests potential receptor binding sites on the underside of the platform on the side opposite of the surface recognized by alphabeta T cell receptors on MHC class I-peptide complexes.
Because the ionized species is more polar than its un-ionized counterpart, it is often assumed that the ionized species of the drug does not make a meaningful contribution to solubilization by either cosolvents or surfactants. This report extends previous studies on solubilization of the ionic species by a combination of pH control and complexation to pH control and micellization and to pH control and cosolvency. The total aqueous solubility is expressed as the addition of the concentration of all contributing species: free un-ionized drug [Du], free ionized drug [Di], un-ionized drug micelle [DuM], and ionized drug micelle [DiM] for surfactant, and free un-ionized drug [Dcu] and free ionized drug [Dci] for cosolvent. The equations indicate that under certain conditions the ionized species can be more important in determining the drug total solubility than the un-ionized species. Flavopiridol, a weak base, is used to test these newly generated equations. As expected, the micellar partition coefficient and solubilization power for ionized flavopiridol are both less than those of the un-ionized species. However, at acidic pH, the solubilities of the ionized drug in surfactant micelles [DiM] and in cosolvent-water [Dci] are both much greater than that of the un-ionized drug. This difference is because the solubilization of the ionized drug is proportional to its aqueous solubility, and its solubility [Di] can be as much as 24-fold greater than that of the free un-ionized species [Du].
This study investigates the roles of both ionized and un-ionized species of flavopiridol in solubilization by complexation, micellization, and cosolvency. Control of pH was used in combination with surfactants (polysorbate 20 and polysorbate 80), cosolvents (ethanol and propylene glycol), as well as uncharged and anionic complexing agents [hydroxypropyl beta-cyclodextrin (HPbetaCD) and sulfobutyl ether beta-cyclodextrin (SBEbetaCD)] to solubilize flavopiridol. These combined techniques increase not only the solubility of the un-ionized flavopiridol but also the solubility of the ionized drug. This study confirms that previously developed equations effectively characterize the roles of pH, pK(a), and either complexation constant, micelle partition coefficient, or cosolvent solubilizing power in determining drug total aqueous solubility.