Biomedical subjects

N Ling

Publications and source records attributed to N Ling.

At least 37 records · Page 2Linked to original sources

PubMed1999

Genetic variation, population structure and cryptic species within the black mudfish, Neochanna diversus, an endemic galaxiid from New Zealand.

To investigate the phylogenetic relationships and geographical structure among landlocked populations of the black mudfish, Neochanna diversus, mitochondrial DNA nucleotide sequence data were sampled from seven populations from the Waikato and Northland regions of New Zealand. The complete D-loop region was sequenced from 70 individuals, with 913 bp from the tRNA-pro end used in population and phylogenetic analysis. A tandem repeat array, which ranged in size up to 200 bp, was found in most populations at the 3' end of the D-loop that was not able to be aligned for analysis. Of the seven sites sampled, two from Northland exhibited significant sequence divergence from all other sites. There was also a clear distinction among remaining Northland sites and those from the Waikato. An additional 518 bp segment of the 16S region was sequenced from all sites and compared with the other New Zealand mudfish species, N. apoda, N. burrowsius and the Tasmanian mudfish Galaxias (Neochanna) cleaveri using Galaxias maculatus as an outgroup. Both D-loop and 16S sequence data provided strong evidence for a cryptic species of mudfish present in Northland. The significant genetic structure apparent in the black mudfish appears most probably to be attributed to geological conditions during the Pliocene, where peat wetlands became apparent in the Waikato while Northland consisted of disjunct 'islands'. Conservation and management of these populations must take into account the historical processes that have shaped these patterns of genetic diversity.

Animals↗

PubMed1998

Characterization of [125I-Tyr0]-corticotropin releasing factor (CRF) binding to the CRF binding protein using a scintillation proximity assay.

We describe the characterization of high affinity [125I-Tyr0]-human CRF binding to purified recombinant human CRF-binding protein (CRF-BP) using a scintillation proximity assay (SPA). For this stable nonseparation technique developed in 96 well microtiter plates, biotinylated CRF-BP is captured by streptavidin-coated SPA beads for the detection of bound [125I-Tyr0]-CRF. Unbound [125I-Tyr0]-CRF represented little or no signal in the assay. Total binding observed was greater than 5000 cpm with a nonspecific signal of < 100 cpm determined in the presence of excess unlabeled human CRF. A comparison of the SPA method with a charcoal precipitation method confirmed that the biotinylation procedure did not adversely affect affinity of the CRF-BP for [125I-Tyr0]-CRF. Saturation binding analysis yielded an apparent equilibrium dissociation constant (Kd) of 208 +/- 5.0 pM (+/- S.D., n = 3). An inhibition constant (Ki) for unlabeled CRF was calculated to be 0.22 +/- 0.03 nM (+/- S.D., n = 8) and a pharmacological profile for eight CRF-related neuropeptides gave a rank potency similar to previously reported results. Finally, the assay variability was assessed with intra- and inter-plate coefficients of variation which were less than 5% each.

Animals↗

PubMed1998

Short peptide-based tolerogens without self-antigenic or pathogenic activity reverse autoimmune disease.

An immunodominant epitope of myelin basic protein (MBP), VHFFKNIVTPRTP (p87-99), is a major target of T cells in brain lesions of multiple sclerosis (MS), and this peptide can trigger experimental autoimmune encephalomyelitis (EAE). We designed truncated peptides based on this pathogenic 13-mer that are not antigenic. These short peptides reduced production of IFN-gamma and TNF-alpha in vivo. Moreover, paraplegic rats given the 7-mer FKNIVTP in soluble form showed total reversal of paralysis in 24 h. Truncated peptides that are too small to stimulate antigenic responses to pathogenic regions of myelin basic protein are nevertheless effective tolerogens and are able to anergize autoreactive T cells. Short peptide-based tolerogens, devoid of immunogenic and pathogenic potential, may be attractive for therapy of autoimmune diseases.

Animals↗

PubMed1998

Predictable TCR antigen recognition based on peptide scans leads to the identification of agonist ligands with no sequence homology.

The potential of CD4+ T cells for cross-recognition of self and foreign Ags has important implications for the understanding of thymic selection, lymphocyte survival, and the occurrence of autoimmune diseases. Here, we define the extensive flexibility of Ag recognition for three human CD4+ autoreactive T cell clones (TCC) by using ligands with single and multiple amino acid (aa) substitutions. Our results demonstrate that the spectrum of tolerated ligands and the resulting stimulatory potency of peptides for a TCC can be predicted by the relative influence of each aa. Using this approach, we have identified stimulatory ligands not sharing a single aa in corresponding positions with the Ag used to establish the TCC. These results argue for an independent contribution of each aa in the peptide sequence to the affinity of the MHC/peptide complex to the TCR.

Amino Acid Sequence↗

PubMed1998

Displacement of insulin-like growth factors from their binding proteins as a potential treatment for stroke.

Insulin-like growth factors I and II (IGF-I and IGF-II) play an important role in normal growth and brain development and protect brain cells from several forms of injury. The effects of IGFs are mediated by type-I and type-II receptors and modulated by potentially six specific binding proteins that form high-affinity complexes with IGFs in blood and cerebrospinal fluid (CSF) and under most circumstances inactivate them. Because brain injury is commonly associated with increases in IGFs and their associated binding proteins, we hypothesized that displacement of this large "pool" of endogenous IGF from the binding proteins would elevate "free" IGF levels to elicit neuroprotective effects comparable to those produced by administration of exogenous IGF. A human IGF-I analog [(Leu24, 59, 60, Ala31)hIGF-I] with high affinity to IGF-binding proteins (Ki = 0.3-3.9 nM) and no biological activity at the IGF receptors (Ki = >10,000 nM) increased the levels of "free, bioavailable" IGF-I in the CSF. Intracerebroventricular administration of this analog up to 1h after an ischemic insult to the rat brain had a potent neuroprotective action comparable to IGF-I. This novel strategy for increasing "free" IGF levels in the brain may be useful for the treatment of stroke and other neurodegenerative diseases.

Animals↗

PubMed1998

Interferon beta 1-b in verbal memory functioning of patients with relapsing-remitting multiple sclerosis.

The effects of interferon Beta 1-b (Betaseron) on verbal memory functioning was examined in 167 patients with relapsing-remitting multiple sclerosis and 112 matched normal controls. Subjects were administered 10 verbal memory tests from the Memory Assessment Scales and the Verbal subtests from the Wechsler Adult Intelligence Scale. Analysis showed subjects treated with Betaseron (n = 73) did not perform significantly better on measures of verbal memory or verbal ability than subjects not receiving the drug (n = 94), although the mean performance of treated subjects was higher across all verbal memory tests. Both groups of patients performed significantly worse on verbal memory subtests measuring list acquisition, delayed list recall, delayed cued recall, and the immediate and delayed recall of names and faces than control subjects. Although patients had lower performance scores across all memory tests than the control subjects, their scores were not within the impaired range. These results do not permit a clear conclusion about the effects of Betaseron on verbal memory for any effect is probably obscured by the relatively preserved cognitive functioning of this outpatient sample.

Adjuvants, Immunologic↗

PubMed1997

Systemic therapy with 3BIT, a triple combination cocktail of anti-CD19, -CD22, and -CD38-saporin immunotoxins, is curative of human B-cell lymphoma in severe combined immunodeficient mice.

We demonstrate in these preclinical studies that all severe combined immunodeficient mice injected with the human B-cell lymphoma cell line Ramos are cured when treated with a combination of anti-CD19, -CD22, and -CD38-saporin immunotoxins (ITs; termed 3BIT). Each component IT used individually did not cure the majority of animals but did significantly prolong their survival compared with PBS sham-treated controls, although the majority succumbed eventually to disease. The very significant improvement obtained with the three-IT combination 3BIT was not due to an antibody or antibody-plus-IT effect. We postulate that by targeting against these three cell surface molecules, we have effectively ensured delivery of saporin to each lymphoma cell with growth potential within the tumor, thus overcoming the problems of heterogeneity of target antigen expression that can limit the therapeutic efficacy of single-IT therapy or even two-IT combination therapy. These "proof of principle" findings have an obvious important bearing on antibody-based therapies for cancer and provide the rationale needed for the design and implementation of clinical trials with such combinations.

ADP-ribosyl Cyclase↗

PubMed1997

Stress and autoimmunity: the neuropeptides corticotropin-releasing factor and urocortin suppress encephalomyelitis via effects on both the hypothalamic-pituitary-adrenal axis and the immune system.

Corticotropin-releasing factor (CRF) exerts a major role in the stress response. Both CRF and urocortin, a newly discovered neuropeptide homologous to CRF, suppressed experimental autoimmune encephalomyelitis (EAE). Suppression of paralysis with CRF involved stimulation of the hypothalamic-pituitary-adrenal axis and inhibitory effects on an encephalitogenic T cell line. While CRF increased glucocorticoid production, which is known to block EAE, it also suppressed EAE in adrenalectomized rats, where glucocorticoid stimulation via CRF plays no role. Moreover, the encephalitogenicity of a T cell line exposed to CRF in vitro was reduced. Stress may influence autoimmune disease through the hypothalamic-pituitary-adrenal axis and directly via the immune system.

Animals↗

PubMed1997

Modifications of peptide ligands enhancing T cell responsiveness imply large numbers of stimulatory ligands for autoreactive T cells.

In this report, we demonstrate for autoreactive T cell clones that single amino acid modifications of the antigenic ligand can result in not only abrogated, decreased, or unmodified, but also increased, T cell responsiveness (superagonist ligands). We further studied the effects of combinations of multiple substitutions with different effects in single peptides. Experiments with peptides carrying multiple amino acid exchanges revealed that the final outcome of TCR ligation by a given ligand is the integration of negative, neutral, and positive effects of each single residue. In addition, the introduction of superagonist substitutions together with nonconservative modifications of primary and secondary TCR contacts resulted in stimulatory ligands. These findings indicate that: 1) the specificity of a single TCR is highly degenerate; 2) ligands exist for autoreactive T cells that have higher agonist activity than the autoantigen itself; 3) the rules to search for cross-reactive epitopes in autoimmunity should take into account that amino acids at certain positions within an antigenic peptide may exert superagonist activity and compensate for the negative effects of residues at other positions that would otherwise not be tolerated.

Amino Acid Sequence↗

PubMed1997

Immunosuppressive phenotype of corticotropin-releasing factor transgenic mice is reversed by adrenalectomy.

Stress elicits a wide range of physiological changes involving the nervous, endocrine, and immune systems. Corticotropin-releasing factor (CRF) plays a key role in orchestrating this response, activating both the sympathetic nervous system and the hypothalamic-pituitary-adrenal axis, resulting in release of corticosteroids. The present study examines the immunological phenotype and responsiveness of CRF-transgenic (CRF-Tg) mice. The immune system of the CRF-Tg animals has profound changes compared to littermate controls, including a marked reduction in both cell number and immune responsiveness. There were also phenotypic changes in the lymphocytic composition of the various lymphoid organs, most notably in the spleen, where CRF-Tg mice had a greater percentage of T lymphocytes compared to littermate controls. Adrenalectomy of CRF-Tg reversed the immunological phenotype observed and restored immune responsiveness. These results demonstrate that CRF overexpression leads to profound impairment on lymphocyte development and function mediated via corticosteroids.

Adrenalectomy↗

PubMed1997

Amelioration of relapsing experimental autoimmune encephalomyelitis with altered myelin basic protein peptides involves different cellular mechanisms.

T-cells specific for a region of human myelin basic protein, amino acids 87-99 (hMBP87-99), have been implicated in the development of multiple sclerosis (MS) a demyelinating disease of the central nervous system. Administration of soluble altered peptide ligand (APL), made by substituting native residues with alanine at either positions 91(91K > A or A91) or 97 (97R > A or A97) in the hMBP87-99 peptide, blocked the development of chronic relapsing experimental autoimmune encephalomyelitis (R-EAE), in the SJL mouse. The non-encephalitogenic APL A91, appears to induce cytokine shifts from Th1 to Th2 in the target T-cells, whereas the encephalitogenic superagonist APL A97 causes deletion of the MBP87-99 responsive cells. Thus, single amino acid changes at different positions in the same peptide epitope can lead to APL capable of controlling auto-immune disease by different mechanisms.

Animals↗

PubMed1997

Enhancement of performance in multiple learning tasks by corticotropin-releasing factor-binding protein ligand inhibitors.

Evidence favors a role for corticotropin-releasing factor (CRF) in learning and memory processes. A binding protein (CRF-BP) with the ability to inactivate CRF provides a novel target to modulate endogenous levels of CRF. The present studies employed three measures of information processing in rats in order to examine the impact of CRF system activation resulting from administration of CRF-BP ligand inhibitors, which increase levels of "free CRF." Acquisition of a visual discrimination paradigm and retention of a inhibitory avoidance task were dose dependently facilitated by central administration of a CRF-BP ligand inhibitor. CRF-BP ligand inhibitor treatment also improved performance in an active avoidance paradigm in aged animals. No nonspecific anorexic effects of the active dose of CRF-BP ligand inhibitor were detected in a food intake test. Moreover, the magnitude of in vivo efficacy of the CRF-BP ligand inhibitor peptide in producing a mild increase in motor activity was dissociated from that of a postsynaptic CRF receptor agonist that exerted robust and long-lasting activity increases. Thus, CRF-BP ligand inhibitors appear to elicit generalized learning enhancement effects without mimicking the robust nonspecific behavioral actions of a CRF receptor agonist.

Animals↗

PubMed1997

Expression and characterization of a putative high affinity human soluble leptin receptor.

Leptin, a circulating 16-kDa protein secreted by adipocytes, decreases body weight by reducing food intake and enhancing energy utilization. Leptin receptors that share homology to the glycoprotein gp130 have been recently cloned. In addition, differentially spliced leptin receptor messenger RNAs have been identified. Functional mutations in either the leptin or leptin receptor gene cause obesity. In the present study, expression of the full length human leptin receptor complementary DNA encoding the long cytoplasmic domain of leptin receptor in COS7 cells resulted in high affinity membrane binding of 125I-leptin (Ki approximately 200 pM); no detectable binding was present in the medium. In addition, we expressed the extracellular domain of human leptin receptor in COS7 cells and identified a soluble leptin receptor in the conditioned medium that binds human and mouse leptin with high affinity comparable with the full length membrane receptor. Transfected COS7 cells expressing the soluble leptin receptor also demonstrated modest specific 125I-leptin binding in whole cells, presumably due to association of the soluble leptin receptor to cell membrane proteins. Data from cross-linking studies identified two specific bands in the 125I-leptin/soluble leptin receptor complex with molecular masses of approximately 130-150 kDa and 300 kDa. The 130-150 kDa molecular mass was confirmed in Western blot analysis and Coomassie staining of the purified soluble receptor and probably represents the glycosylated form of the receptor. The 300-kDa band most likely represents a homodimer of the soluble leptin receptor complex because HPLC gel filtration analysis of the 125I-leptin/soluble leptin receptor complex identified a single peak corresponding to a molecular mass of approximately 340 kDa. The soluble leptin receptor antagonized 125I-leptin binding to the membrane receptor, suggesting its potential utility as a functional tool for determining the role of endogenous leptin.

Animals↗

PubMed1997

Human T-cell response to myelin basic protein peptide (83-99): extensive heterogeneity in antigen recognition, function, and phenotype.

Multiple sclerosis (MS) is considered a T cell-mediated autoimmune disease, and myelin proteins are the most likely candidate autoantigens. Based on experiments performed in experimental allergic encephalomyelitis (EAE), innovative immunotherapies have been developed that target either the specific trimolecular complex of encephalitogenic T cells, consisting of T-cell receptor (TCR), major histocompatibility complex (MHC; HLA in humans) class II molecule, and autoantigenic peptide, or the effector functions of these cells. To provide the basis for the transfer of these specific immunotherapies to MS, we extensively characterized the human T-cell response to one major myelin epitope, the myelin basic protein peptide (83-99). We analyzed restriction element, TCR usage and affinity, fine specificity, cytokine production, cytolytic activity, and expression of surface molecules on 41 T-cell clones (TCCs) derived from MS patients and normal controls. We demonstrate a high degree of complexity of recognition patterns as well as of functional phenotypes among T cells responding to the same epitope. In contrast to results from animal models, these findings indicate that the design of epitope-based specific immunotherapies for MS is more difficult than previously thought.

Antigens↗

PubMed1997

[Studies on chemical constituents of Polygala arillata buch-ham].

Two new compounds were isolated from the roots of Polygala arillata Buch-Ham. On the basis of chemical reactions and spectral (UV, IR, MS, 1HNMR, DIFNOE, 13CNMR) analysis, they were identified as 1,3-dihydroxy-2-methoxyxanthone(I) and 7-hydroxy-1-methoxy-2,3-methylenedioxyxanthone(II). Pharmacological study indicated that I and II have inhibitory effect on aldose reductase activity.

Aldehyde Reductase↗

PubMed1996

Corticotropin-releasing factor-binding protein ligand inhibitor blunts excessive weight gain in genetically obese Zucker rats and rats during nicotine withdrawal.

Elevation of the neuropeptide corticotropin-releasing factor (CRF) in the brain is associated with a reduction of food intake and body weight gain in normal and obese animals. A protein that binds CRF and the related peptide, urocortin, with high affinity, CRF-binding protein (CRF-BP), may play a role in energy homeostasis by inactivating members of this peptide family in ingestive and metabolic regulatory brain regions. Intracerebroventricular administration in rats of the high-affinity CRF-BP ligand inhibitor, rat/human CRF (6-33), which dissociates CRF or urocortin from CRF-BP and increases endogenous brain levels of "free" CRF or urocortin significantly blunted exaggerated weight gain in Zucker obese subjects and in animals withdrawn from chronic nicotine. Chronic administration of CRF suppressed weight gain nonselectively by 60% in both Zucker obese and lean control rats, whereas CRF-BP ligand inhibitor treatment significantly reduced weight gain in obese subjects, without altering weight gain in lean control subjects. Nicotine abstinent subjects, but not nicotine-naive controls, experienced a 35% appetite suppression and a 25% weight gain reduction following acute and chronic administration, respectively, of CRF-BP ligand inhibitor. In marked contrast to the effects of a CRF-receptor agonist, the CRF-BP ligand inhibitor did not stimulate adrenocorticotropic hormone secretion or elevate heart rate and blood pressure. These results provide support for the hypothesis that the CRF-BP may function within the brain to limit selected actions of CRF and/or urocortin. Furthermore, CRF-BP may represent a novel and functionally selective target for the symptomatic treatment of excessive weight gain associated with obesity of multiple etiology.

Animals↗

PubMed1996

Urocortin interaction with corticotropin-releasing factor (CRF) binding protein (CRF-BP): a novel mechanism for elevating "free' CRF levels in human brain.

Here we demonstrate that urocortin, a new mammalian member of the corticotropin-releasing factor (CRF) neuropeptide family has high affinity for both the recombinant human CRF binding protein (CRF-BP) and for a membrane-associated form of the protein solubilized from postmortem human cerebrocortical brain tissue. The rank order of binding potency for both the human recombinant CRF-BP and for the solubilized human brain CRF-BP is: urotensin > hCRF > urocortin > sauvagine. The bound hCRF/hCRF-BP complex was detected in the postmortem human brain tissue using an ELISA assay specific for the hCRF/hCRF-BP complex. A large proportion (65%) of the endogenous hCRF was found to be complexed to the CRF-BP and thus unavailable for CRF receptor activation. Incubation of human brain postmortem tissue extracts with urocortin and urotensin resulted in a dramatic decrease in hCRF/hCRF-BP levels and a concomitant increase in "free' hCRF levels. Thus, urocortin and other putative CRF-related peptides may elevate endogenous levels of "free' hCRF in brain by displacing hCRF from the binding protein. These data define an indirect endogenous mechanism for activation of CRF receptors by new mammalian members of the CRF family of neuropeptides.

Binding Sites↗

PubMed1996

Treatment of experimental encephalomyelitis with a peptide analogue of myelin basic protein.

Following induction of experimental encephalomyelitis with a T-cell clone, L10C1, that is specific for the myelin basic protein epitope p87-99, the inflammatory infiltrate in the central nervous system contains a diverse collection of T cells with heterogeneous receptors. We show here that when clone L10C1 is tolerized in vivo with an analogue of p87-99, established paralysis is reversed, inflammatory infiltrates regress, and the heterogeneous T-cell infiltrate disappears from the brain, with only the T-cell clones that incited disease remaining in the original lesions. We found that antibody raised against interleukin-4 reversed the tolerance induced by the altered peptide ligand. Treatment with this altered peptide ligand selectively silences pathogenic T cells and actively signals for the efflux of other T cells recruited to the site of disease as a result of the production of interleukin-4 and the reduction of tumour-necrosis factor-alpha in the lesion.

Amino Acid Sequence↗