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Biomedical subjects

G Ju

Publications and source records attributed to G Ju.

At least 73 records · Page 4Linked to original sources
PubMed1997

[Effect of acetylcholine receptor antibody derived from myasthenia gravis on the rat central neuronal nicotinic acetylcholine receptor].

Immunoreaction between acetylcholine receptor antibody (AChRab) derived from myasthenia gravis (MG) and neuronal nicotinic acetylcholine receptor (AChR) and the action of AChRab on rat brain stem auditory transmission were investigated by immunocytochemistry and brain stem auditory evoked potential (BAEP) in a search for abnormalities indicating central nervous system (CNS) involvement in this disorder. 25 microliters of AChRab were injected into the ventricular system of rats once every 48 hours for periods of up to a week BAEP examination was taken before and after injection of AChRab. Immunocytochemical investigation of the rat brain sections provided evidence that the neuronal nicotinic AChR like immunoreactivity is widely distributed throughout the rat CNS such as cerebral cortex, hippocampus, motor neurons in brain stem, auditory nucleus cerebellar cortex, etc. Analysis of the immunocytochemical results showed that the distribution property of AChRab in rat CNS correlated well with known neuronal nicotinic AChR subtypes e.g., alpha 7 and alpha 4. BAEP inter peak latency was significantly prolonged in rats after injection of AChRab as compared with the control rats and the rats prior to injection of AChRab. The results indicated that cerebroventricular administration of AChRab not only cause dysfunction of brain stem auditory transmission but also leads to development of experimental autoimmune MG symptoms. It is concluded that AChRab influences the rat brain stem auditory transmission and it is suggested that the pathogenic effect of AChRab is not only limited at neuromuscular junction, but also involves the central neuronal nicotinic AChR.

Animals↗
PubMed1997

[Whole-cell recordings of the supraoptic nucleus neurons from rat hypothalamic slices in vitro].

Fifty-two supraoptic nucleus neurons in rat slice preparations were studied using whole cell patch-clamp technique. The mean passive and active membrane properties were measured as follows: resting membrane potential, 59 +/- 8 mV; input resistance, 535 +/- 129 M omega; time constant, 32 +/- 9 ms; amplitude of the action potentials, 99 +/- 11 mV; overshoot, 37 +/- 13 mV (n = 39). Most of these neurons showed a prominent slow after-hyperpolarization potential or current in response to depolarizing pulse. In votage-clamp condition, it was found that virtually all supraoptic neurons (n = 13) were invaded by spontaneous synaptic inputs. Pharmacological experiments showed that the excitatory postsynaptic currents (EPSCs) were mediated by non-NMDA glutamate receptors, whereas inhibitory postsynaptic currents (IPSCs) by GABAA receptors.

Animals↗
PubMed1996

Fos oncoprotein expression in the rat forebrain following muscimol-induced absence seizures.

Fos oncoprotein expression is a marker of neuronal activation following seizures. Here, using this method we examined the anatomical locations of muscimol-induced absence seizures in the rat forebrain. Six hours after a systemic injection of muscimol a massive Fos immunoreactivity appeared in the olfactory system, retrosplenial cortex and paraventricular thalamic nucleus, whereas other cortical areas contained low level of Fos expression. These results provide the first functional morphological evidence suggesting that these forebrain structures with Fos expression may play an important role in the pathophysiology of muscimol-induced absence seizures.

Animals↗
PubMed1996

Synaptic relationship of substance P-like-immunoreactive nerve fibers with gland cells of the anterior pituitary in the rat.

Our previous studies have proved the presence of substantial amounts of peptidergic nerve fibers in the anterior pituitary of human, monkey, dog, and rat, and synaptic contacts have been demonstrated on corticotrophs and somatotrophs in the dog. The present study is aimed at investigating the synaptic relationship between axons and gland cells in the rat. The substance P-like-immunoreactive nerve fibers were studied immuno-electron-microscopically. They were nonmyelinated and varicose. Typical synaptic contacts between the fibers and every type of the hormone-secreting gland cell could be identified, most frequently on lactotrophs. No synaptic contacts were found on the folliculo-stellate cell. The synaptic contacts were of asymmetrical type with clusters of small round clear vesicles at the presynaptic site and scattered large dense-cored vesicles. This pattern of synaptic relationships was markedly different from that in the dog. The presence of synapses in the anterior pituitary of the rat implies a direct neural regulation of certain cells and strongly supports our hypothesis of neural-humoral dual regulation of this gland.

Animals↗
PubMed1995

Distinction between gamma c detection and function in YT lymphoid cells and in the granulocyte-macrophage colony-stimulating factor-responsive human myeloid cell line, Tf-1.

Peripheral blood monocytes respond to interleukin-2 (lL-2) and express the gamma common (gamma c) subunit of the lL-2 receptor (lL-2R) complex. However, the role of lL-2 in myeloid development has recently become of interest for several reasons, including the effect gamma c mutations may or may not have on myeloid development in patients with XSCID. Many studies of lL-2 function in the myeloid cell lineage have been performed on a murine background. To study gamma c expression and function in human myeloid precursors, we introduced the human myelomonocytic cell line, Tf-1, with a retroviral vector containing the human lL-2R beta subunit to create functional human intermediate lL-2R consisting of beta gamma c dimers. We have characterized this transfected variant of Tf-1 (Tf-1 beta) with regard to its response to lL-2. Unlike the parental Tf-1 cell line that is deficient in both lL-2R alpha and lL-2R beta expression, the Tf-1 beta transfectant binds and responds to lL-2 through intermediate-affinity lL-2Rs. Scatchard analyses indicate the number of intermediate-affinity receptors on Tf-1 beta is similar to the number found on the well-characterized YT cell line. However, detection of gamma c on Tf-1 beta cells is dramatically less than on YT cells by Western blot analysis and is undetectable by flow cytometric studies and surface iodinations. The gamma c component on YT cells is readily detected by all three methods. We conclude from these studies that the intermediate-affinity lL-2Rs on the Tf-1 cell line behave differently than those on YT cells with respect to gamma c detection. Either the gamma c molecule itself is different, or the cellular environment in which it functions is altered. Elucidation of gamma c function on this cell line will allow for its use as a model in which other cytokines using gamma c (including lL-2, lL-4, and lL-15) can be studied on the same cellular background.

Animals↗
PubMed1995

Insertion of a structural domain of interleukin (IL)-1 beta confers agonist activity to the IL-1 receptor antagonist. Implications for IL-1 bioactivity.

We showed previously that replacement of Lys-145 in the IL-1 receptor antagonist (IL-1ra) with Asp resulted in an analog (IL-1ra K145D) with partial agonist activity. To identify additional amino acids that affect IL-1 bioactivity, we created second site mutations in IL-1ra K145D. Substitutions of single amino acids surrounding position 145 were made; none of these substitutions increased the bioactivity of IL-1ra K145D. However, the insertion of the beta-bulge (QGEESN) of IL-1 beta at the corresponding region of IL-1ra K145D resulted in a 3-4-fold augmentation of bioactivity. An additional increase in agonist activity was observed when the beta-bulge was co-expressed with a second substitution (His-54 --> Pro) in IL-1ra K145D. We also show that the bioactivity of both IL-1ra K145D and the triple mutant IL-1ra K145D/H54P/QGEESN is dependent on interaction with the newly cloned IL-1 receptor accessory protein.

Amino Acid Sequence↗
PubMed1995

Molecular cloning and characterization of a second subunit of the interleukin 1 receptor complex.

A monoclonal antibody (mAb) was isolated that blocked the binding and bioactivity of both human and murine interleukin 1 beta (IL-1 beta) on murine IL-1 receptor-bearing cells. This mAb recognized a protein that was distinct from the Type I and Type II IL-1 receptors, suggesting that an additional protein exists that is involved in IL-1 biological responses. By expression cloning in COS-7 cells, we have isolated a cDNA from mouse 3T3-LI cells encoding this putative auxiliary molecule, which we term the IL-1 receptor accessory protein (IL-1R AcP). Sequence analysis of the cDNA predicts an open reading frame that encodes a 570-amino acid protein with a molecular mass of approximately 66 kDa. The IL-1R AcP is a member of the Ig superfamily by analysis of its putative extracellular domain and also bears limited homology throughout the protein to both Type I and Type II IL-1 receptors. Northern analysis reveals that murine IL-1R AcP mRNA is expressed in many tissues and appears to be regulated by IL-1. In mammalian cells expressing natural or recombinant Type I IL-1R and IL-1R AcP, the accessory protein forms a complex with the Type I IL-1R and either IL-1 alpha or IL-1 beta but not IL-1ra. The recombinant accessory protein also increases the binding affinity of the recombinant Type I IL-1R for IL-1 beta when the two receptor proteins are coexpressed. Therefore, the functional IL-1 receptor appears to be a complex composed of at least two subunits.

3T3 Cells↗
PubMed1995

Neuropeptide Y and galanin binding sites in rat and monkey lumbar dorsal root ganglia and spinal cord and effect of peripheral axotomy.

Using monoiodinated peptide YY (PYY) and galanin as radioligands, and neuropeptide Y (NPY) fragments, the distribution of NPY binding sites and its subtypes Y1 and Y2, and of galanin binding sites, was investigated in rat and monkey lumbar (L) 4 and L5 dorsal root ganglia (DRG) and spinal cord before and after a unilateral sciatic nerve cut, ligation or crush. Receptor autoradiography revealed that [125I]PYY bound to some DRG neurons and a few nerve fibres in normal rat DRG, and most of these neurons were small. NPY binding sites were observed in laminae I-IV and X of the rat dorsal horn and in the lateral spinal nucleus, with the highest density in laminae I-II. [125I]PYY binding was most strongly attenuated by NPY13-36, a Y2 agonist, and partially inhibited by [Leu31,Pro34]NPY, a Y1 agonist, in both rat DRG and the dorsal horn of the spinal cord. These findings suggest that Y2 receptors are the main NPY receptors in rat DRG and dorsal horn, but also that Y1 receptors exist. After sciatic nerve cut, PYY binding markedly increased in nerve fibres and neurons in DRG, especially in large neuron profiles, and in laminae III-IV of the dorsal horn, as well as in nerve fibres in dorsal roots and the sciatic nerve. Incubation with NPY13-36 completely abolished PYY binding, which was also reduced by [Leu31,Pro34] NPY. However, the increase in PYY binding seen in laminae I-IV of the ipsilateral dorsal horn after axotomy was not observed after coincubation with [Leu31,Pro34] NPY. NPY binding sites were seen in a few neurons in monkey DRG and in laminae I-II, X and IX of the monkey spinal cord. The intensity of PYY binding in laminae I-II of the dorsal horn was decreased after axotomy. Galanin receptor binding sites were not observed in rat DRG, but were observed in the superficial dorsal horn of the spinal cord, mainly in laminae I-II. Axotomy had no effect on galanin binding in rat DRG and dorsal horn. However, galanin receptor binding was observed in many neurons in monkey L4 and L5 DRG and in laminae I-IV and X of monkey L4 and L5 spinal cord, with the highest intensity in laminae I-II. No marked effect of axotomy was observed on the distribution and intensity of galanin binding in monkey DRG or spinal cord.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1995

Site-directed mutagenesis--molecular biology and rational drug design.

Using site-directed mutagenesis, we have determined the location and composition of the binding sites in human IL-1 alpha and IL-1 beta for the Type I IL-1 receptor (IL-1R). The binding site in each ligand is a discontinuous epitope made up of at least seven amino acids whose side chains are exposed on a contiguous region of the protein surface. Although human IL-1 alpha and IL-1 beta have similar affinities and cross-compete for binding to the human Type I IL-1R, the binding site residues are not identical in the two ligands. In addition, the residues in the binding site of each ligand contribute differently to binding of the human versus the mouse IL-1R. The structure of the IL-1 binding site has implications for the rational design of IL-1 antagonists.

Animals↗
PubMed1995

The parabrachio-subfornical organ projection in the rat.

Previous physiological studies have shown that both the parabrachial nucleus and the subfornical organ are involved in drinking behavior and cardiovascular controls. The purpose of the present work was to study the direct connections between these two structures by using anterograde and retrograde transport methods. A mixture of wheat germ agglutinin conjugated with horseradish peroxidase and free horseradish peroxidase or Fluorogold was injected into either the parabrachial nucleius (PBN) or the subfornical organ (SFO). The results indicated that the parabrachial nucleus sends a substantial projection to the entirety of the subfornical organ, and this input appears to be distributed to both the central and peripheral regions of this structure. Neurons that give origin to this projection are mainly located in the outer layer of the lateral division of the parabrachial nucleus, including the superior, internal, dorsal, and external lateral subnuclei. These findings suggest that, besides the already known connections, there is an additional parabrachio-subfornical pathway that may be involved in the central integration of cardiovascular function and drinking behavior.

Animals↗
PubMed1995

Evidence for axonal sprouting in the anterior pituitary following adrenalectomy in the rat.

Our recent studies have demonstrated the presence of peptidergic nerve fibers in the anterior pituitary of the rat. They were found to increase in number following adrenalectomy and the present study was aimed at investigating whether axonal sprouting could account for this increase. Antibody against the neuronal growth-associated protein GAP-43 was used as the probe. Four days following adrenalectomy GAP-43-like immunoreactivity was found to increase dramatically, mostly as varicosities surrounding the gland cells. The results suggest an active axonal sprouting following this hormone manipulation and strongly support our hypothesis of neural-humoral dual regulation of the mammalian anterior pituitary.

Adrenalectomy↗
PubMed1994

Origin of CCK-like immunoreactive nerve fibers in the neurohypophysis of the rat.

Wheatgerm agglutin (WGA) conjugated horseradish peroxidase (HRP) retrograde tracing technique was combined with immunohistochemistry to study the origin of cholecystokinin (CCK)-like immunoreactive (ir) nerve fibers in the neurohypophysis of the rat. The CCK-ir neurons projecting to the neurohypophysis were localized mainly in the magnocellular paraventricular nuclei (mPVN), periventricular PVN, medial parvocelullar PVN, medial preoptic area and dorsal accessory nuclei in region between the stria medullaris and fornix. Double-labeled cells were also observed in small amounts in the dorsal parts of supraoptic nuclei, the ventral parts of anterior hypothalamic regions and lateral hypothalamic areas. In addition, a few double-labeled cells were seen in the subependymal area of the interventricular foramen (IVF) and in the floor of the 3rd ventricle. Our results demonstrate that CCK-ir nerve fibers in the posterior pituitary originate from more widespread hypothalamic areas than generally assumed. The fact that some CCK-ir neurons projecting to the posterior pituitary were located close to cerebrospinal fluid suggests that they may play a role in monitoring and controlling changes of cerebrospinal fluid.

Animals↗
PubMed1994

Cooperative interactions between the interleukin 2 receptor alpha and beta chains alter the interleukin 2-binding affinity of the receptor subunits.

The interleukin 2 (IL-2) receptor (IL-2R) is a multisubunit receptor that includes three major IL-2 binding subunits, the IL-2R alpha, beta, and gamma chains. We have detected and analyzed cooperative interactions between the IL-2R alpha and beta chains (IL-2R alpha and IL-2R beta, respectively) in COS cells transfected with cDNAs encoding the IL-2R alpha, the IL-2R beta, or both cDNAs. We demonstrated that IL-2 F42A, an analog that fails to bind to the isolated IL-2R alpha subunit and would be predicted by the hierarchical affinity-conversion model to have impaired binding to cells expressing both chains, instead readily binds to the IL-2R alpha/beta heterodimer in COS cells. Furthermore, this binding is abolished by the antibody HIEI that separates the two IL-2R subunits. The monoclonal antibodies anti-Tac and Mik-beta 1 directed at the IL-2-binding sites on IL-2R alpha and IL-2R beta, respectively, block ligand binding to the heterodimer. This binding pattern is inconsistent with the strict hierarchical affinity-conversion model that mandates an initial binding of IL-2 to IL-2R alpha followed by binding of the IL-2/IL-2R alpha complex to IL-2R beta. Instead, our results support an alternative model of preformed complexes of IL-2R beta with other IL-2R subunits. In this alternative model, IL-2R alpha and -beta exist in part as preformed complexes in which the affinity of IL-2R beta for IL-2 is altered by the proximity of IL-2R alpha, through mechanisms that do not require the prior binding of IL-2 to IL-2R alpha.

Cell Line↗
PubMed1994

Nitric oxide synthase expression in vagal complex following vagotomy in the rat.

Peripheral axotomy of the spinal nerve and avulsion of the ventral roots have been found to induce increase in expression of nitric oxide synthase (NOS) in the spinal motor neurones and the dorsal root ganglion. The present study investigated changes of NOS, using NADPH-diaphorase (NADPH-d) reactivity as the marker, in vagal complex after axotomy in the rat. Eight days after left cervical vagotomy the NADPH-d reactivity was found to be markedly enhanced in the dorsal motor nucleus of the vagus nerve, the ambiguus nucleus, the solitary tract and the nucleus of the tractus solitarius, and the nodose ganglion. This study offers the first evidence of changes in NOS expression in cranial visceral components following axotomy.

Amino Acid Oxidoreductases↗
PubMed1994

Induction of lymphokine-activated killing with reduced secretion of interleukin-1 beta, tumor necrosis factor-alpha, and interferon-gamma by interleukin-2 analogs.

BACKGROUND: Interleukin-2 (IL-2)-based immunotherapy has been shown to effect clinical responses in 15-35% of patients with metastatic renal cell carcinoma or melanoma. Despite its clinical efficacy, many clinicians refrain from using IL-2 because of the associated toxicity. This toxicity is believed to be mediated by such secondary cytokines as IL-1, tumor necrosis factor (TNF), and interferon (IFN)-gamma, which are produced by the patient's IL-2-stimulated peripheral blood mononuclear cells (PBMCs). METHODS: Human PBMCs were stimulated with 1 nM wild-type recombinant IL-2 (rIL-2) or IL-2 analogs (R38A or F42K) that preferentially bind to the intermediate affinity IL-2 receptor (IL-2R). PBMCs were activated for lymphokine-activated killer (LAK) activity in 4-h 51Cr-release assays, using Daudi target cells. Cytokine content in the culture supernatants was determined by enzyme-linked immunosorbent assay. RESULTS: Both R38A and F42K were capable of generating substantial LAK activity. Maximal specific lysis was 54% for PBMCs activated by R38A and 52% for F42K-stimulated cells, in contrast to 64% for rIL-2. In addition, analog-stimulated PBMCs secreted 59% of the IL-1 beta, 25% of the TNF-alpha, and only 8% of the IFN-gamma produced in response to rIL-2 (all p < 0.01 compared with rIL-2-stimulated secretion; one-way ANOVA). CONCLUSIONS: IL-2 analogs that preferentially bind the intermediate-affinity IL-2R retain the capacity to induce substantial LAK activity despite a greatly reduced secondary cytokine production. Therefore, such IL-2 analogs may provide an effective, yet less toxic means of cancer immunotherapy.

Cytotoxicity Tests, Immunologic↗
PubMed1994

Response of calcitonin-gene-related peptide-like immunoreactive nerve fibers of the anterior pituitary to adrenalectomy in the rat.

The mammalian anterior pituitary is known to be regulated by humoral factors only, no direct neural influence having ever been discovered. Our recent studies have shown in several species that there are substantial amounts of substance P and calcitonin-gene-related peptide-like immunoreactive (CGRP-LI) nerve fibers in the anterior pituitary and that they can make synaptic contacts with secretory cells, among which corticotropes. In the present study, we investigated changes in CGRP-LI nerve fibers of the anterior pituitary after bilateral adrenalectomy in the rat. The results show that the number of CGRP-LI nerve fibers increases significantly. In parallel, they cover a wider area of the gland after the operation, suggesting a direct neural influence.

Adrenalectomy↗
PubMed1993

Nitric oxide synthase-like immunoreactivity in lumbar dorsal root ganglia and spinal cord of rat and monkey and effect of peripheral axotomy.

With the immunofluorescence technique, nitric oxide synthase (NOS)-like immunoreactivity (LI) was found in a few medium-sized and small sensory neurons in lumbar (L) 4 and L5 dorsal root ganglia (DRG) of normal rat, and in most of these neurons, NOS-LI coexisted with calcitonin gene-related peptide and sometimes with substance P and galanin. NOS-immunoreactive nerve fibers, terminals and small neurons were also located in the dorsal horn of the segments 4 and 5 of the rat lumbar spinal cord with the highest density in inner lamina II. Many NOS-positive neurons and fibers were seen in the area around the central canal. A sparse network of NOS-immunoreactive nerve fibers was found in the ventral horn. After unilateral sciatic nerve cut in the rat, the number of NOS-positive neurons increased in the ipsilateral L4 and L5 DRGs, mainly in medium and small neurons, but also in some large neurons and very small neurons. NOS-LI could now also be seen in the ipsilateral dorsal roots, and in an increased number of fibers and terminals in both outer and inner lamina II of the ipsilateral dorsal horn. The number of NOS-immunoreactive neurons in lamina II of the ipsilateral dorsal horn was reduced. In the monkey L4 and L5 DRGs, many small neurons were NOS-immunoreactive, but only a few weakly stained nerve fibers and terminals were found in laminae I-IV of the dorsal horn at L4 and L5 lumbar levels. A few NOS-positive neurons were present in lamina X. The number of NOS-immunoreactive neurons was somewhat reduced in DRGs 14 days after peripheral axotomy, but no certain effect was seen in the dorsal horn. These results, together with earlier in situ hybridization studies, demonstrate that axotomy in rat induces a marked upregulation of NOS synthesis in primary sensory neurons, thus suggesting a role for NO in lesioned sensory neurons. In contrast, no such effect was recorded in monkey, perhaps indicating distinct species differences.

Amino Acid Oxidoreductases↗