Kil Lyong Kim

α-Melanocyte-stimulating Hormone Inhibits Lipopolysaccharide-induced Tumor Necrosis Factor-α Production in Leukocytes by Modulating Protein Kinase A, p38 Kinase, and Nuclear Factor κB Signaling Pathways

The neuropeptide α-melanocyte-stimulating hormone (α-MSH) inhibits inflammation by down-regulating the expression of proinflammatory cytokines such as tumor necrosis factor-α (TNF-α) in leukocytes via stimulation of α-MSH cell surface receptors. However, the signaling mechanism of α-MSH action has not yet been clearly elucidated. Here, we have investigated signaling pathways by which α-MSH inhibits lipopolysaccharide (LPS)-induced TNF-α production in leukocytes such as THP-1 cells. We focused on the possible roles of protein kinase A (PKA), p38 kinase, and nuclear factor κB (NFκB) signaling. In THP-1 cells, LPS is known to activate p38 kinase, which in turn activates NFκB to induce TNF-α production. We found that pretreatment of cells with α-MSH blocked LPS-induced p38 kinase and NFκB activation as well as TNF-α production. This response was proportional to α-MSH receptor expression levels, and addition of an α-MSH receptor antagonist abolished the inhibitory effects. In addition, α-MSH treatment activated PKA, and PKA inhibition abrogated the inhibitory effects of α-MSH on p38 kinase activation, NFκB activation, and TNF-α production. Taken together, our results indicate that stimulation of PKA by α-MSH causes inhibition of LPS-induced activation of p38 kinase and NFκB to block TNF-α production.

Effects of the hinge region of cecropin A(1-8)-magainin 2(1-12), a synthetic antimicrobial peptide, on liposomes, bacterial and tumor cells.

A 20-residue hybrid peptide (CA(1-8)-MA(1-12): KWKLFKKIGIGKFLHSAKKF-NH(2)) incorporating 1-8 residues of cecropin A (CA) and 1-12 residues of magainin 2 (MA) has potent antibiotic activity without hemolytic activity. In order to investigate the effects of the flexible hinge sequence, Gly-Ile-Gly of CA(1-8)-MA(1-12) (CA-MA) on antibiotic activity, CA-MA and its three analogues, CA-MA1, CA-MA2 and CA-MA3 were synthesized. The Gly-Ile-Gly sequence of CA-MA was deleted in CA-MA1 and replaced with Pro and Gly-Pro-Gly in CA-MA2 and CA-MA3, respectively. CA-MA1 and CA-MA3 caused a significant decrease in the bactericidal rate against Escherichia coli and Bacillus subtilis and the tumoricidal activity against four different tumor cells, and the PC/PS (4:1, w/w) vesicle-aggregating and disrupting activities. However, CA-MA2 showed a similar bactericidal rate and antitumor, vesicle-aggregating and disrupting activities, as compared with CA-MA. These results suggested that the flexibility or beta-turn induced by Gly-Ile-Gly or Pro in the central part of CA-MA may be important in the electrostatic interaction of the cationic short alpha-helical region in the N-terminus with the cell membrane surface and the hydrophobic interaction of amphipathic alpha-helical region in the C-terminus with the hydrophobic acyl chains in the cell membrane. CA-MA3 exhibited lower activity in antibacterial, antitumor, and vesicle-aggregating and disrupting activities than CA-MA and CA-MA2. This result suggested that the excessive beta-turn structure by Gly-Pro-Gly in CA-MA3 seems to interrupt the ion channel/pore formation on the lipid bilayer. It was concluded that the appropriate flexibility or beta-turn structure provided by the central hinge is responsible for the effective antibiotic activity of the antimicrobial peptides with the helix-hinge-helix structure.

Ganglioside GQ1b improves spatial learning and memory of rats as measured by the Y-maze and the Morris water maze tests

Gangliosides are major components of cell membranes and are particularly enriched in the mammalian brain where they represent the major lipid constituents of the neuronal cell surface. In the central nervous system, gangliosides have a close connection to many neurophysiological functions related to neurogenesis, proliferation, synaptogenesis, and synaptic transmission. The previously reported effect of the tetra-sialoganglioside GQ1b in hippocampal CA1 neurons of brain slices showed that GQ1b enhanced ATP-induced long-term potentiation (LTP). However, there has been no clear evidence of the effects of GQ1b on learning and memory as measured using behavioral test. In the present study, we performed the Y-maze and the Morris water maze (MWM) tests to reveal the effects of GQ1b on spatial learning and memory following intracerebroventricular (ICV) injection of GQ1b. GQ1b-treated rats showed highly increased performance on the Y-maze and the MWM tests without any significant alteration of basal locomotor activity. Therefore, our behavioral data strongly suggest that GQ1b improves spatial learning and memory in rats. Also, these data support the previous finding that GQ1b treatment in hippocampal CA1 neurons of rodent brain slices increased ATP-induced LTP.

Decreased hippocampal cholinergic neurostimulating peptide precursor protein associated with stress exposure in rat brain by proteomic analysis

The stress response alters behavior, autonomic function, and secretion of multiple hormones, including corticotropin‐releasing factor, adrenocorticotropin hormone, and cortisol, through the hypothalamic‐pituitary‐adrenal axis. Constitutive stress responses lead to a number of psychiatric disorders, including depression, posttraumatic stress disorder, Alzheimers disease (AD), and other anxiety disorders through increased stress hormones and other unknown factors. Here, we performed a proteomic analysis of rat brain exposed to restraint stress compared with a nonstress group by using 2D‐DIGE and MALDI‐TOF analysis. Several proteins were identified by peptide mass fingerprint (PMF), including down‐regulated hippocampal cholinergic neurostimulating peptide precursor protein (HCNP‐pp). The current study demonstrates that HCNP‐pp mRNA and protein expression are decreased in rat hippocampus after stress exposure. The level of HCNP‐pp in H19‐7, a rat hippocampal cell line, significantly decreases with dexamethasone treatment, a synthetic glucocorticoid. Thus, this finding suggests that HCNP‐pp expression may decrease in response to stress exposure. Decreased HCNP‐pp from stress exposure may result in lower levels of HCNP that might contribute to a loss of acetylcholine production.

Antifungal mechanism of a cysteine-rich antimicrobial peptide, Ib-AMP1, from Impatiens balsamina against Candida albicans

The antifungal mechanism of a 20-mer peptide, Ib-AMP1, derived from Impatiens balsamina was investigated. The oxidized (disulfide bridged) Ib-AMP1 showed a 4-fold increase in antifungal activity against Aspergillus flavus and Candida albicans than reduced (non-disulfide bridged) Ib-AMP1. Ib-AMP1 had very low activity for phospholipid disruption when compared with cecropin A(1-8)-magainin 2(1-12), a α-helical amphiphatic, antimicrobial peptide. Confocal microscopy showed that Ib-AMP1 binds on cell surface or penetrates into cell membranes. These results suggested that Ib-AMP1 may manifest its antifungal activity against Candida albicans by inhibiting a distinct cellular process rather than ion channel or pore formation in cell membrane.

Design of novel analogue peptides with potent fungicidal but low hemolytic activity based on the cecropin a‐melittin hybrid structure

In order to design synthetic peptides with potent antifungal activity but low cytotoxic activity under physiological conditions, several analogues of the previously reported cecropin A (CA)‐melittin (ME) hybrid peptide, CA(1‐8)‐ME(1‐12), were synthesized. These analogues were designed by analysis of the α‐helical wheel diagram of CA(1‐8)‐ME(1‐12). Antifungal activities were measured by growth inhibition of the yeast Trichosporon beigelii and by hemolytic assay with human red blood cells, respectively. Substitution of Thr for Lys at position 18 and 19 of CA(1‐8)‐ME(1‐12) caused a dramatic reduction in hemolytic activity. Two analogue peptides (analogue I and III) showed more potent antifungal and lower hemolytic activity than the original peptide. To study the antifungal mechanism of these peptides, fluorescence activated flow cytometry and confocal laser scanning microscopy were performed with the most powerful antifungal analogue I peptide designed in the present study. As determined by propidium iodide staining, fungal cells treated with analogue I or melittin showed higher fluorescence intensity than those treated with the weak antifungal peptide, cecropin A. By confocal microscopy the analogue I was detected in the intracellular region as well as the in cell membrane. These facts suggested that the antifungal function of this novel peptide analogue acts by pore formation in the cell membrane.

Determination of the Protective Effects of Neutralizing Anti-Hepatitis B Virus(HBV) Immunoglobulins by Epitope Mapping with Recombinant HBV Surface-Antigen Proteins

Anti‐hepatitis B virus (HBV) surface‐antigen immunoglobulins prepared from human sera are clinical reagents which have been approved for prophylactic treatment in HBV‐exposed persons. The passive immunoprophylaxis with immunoglobulins is meant to cross‐link viral particles, which are then further cleared by the hosts own immune system. While antibodies specific for both anti‐S‐ and anti‐preS proteins have been proved to serve as effective anti‐viral agents, so far the fine antigen specificity of clinical immunoglobulin preparations has not been determined. Using recombinant proteins covering the hepatitis B surface antigen, in the present study, the specificity of a commercially available immunoglobulin preparation was determined and immunodominant epitopes were mapped. Here, it is shown that the major reactivity of anti‐HBV immunoglobulins is directed against the S‐protein, and that no reactivity to the preS2 but a weak binding activity to the preS1 region was detectable. The antigen reactivity within the preS1 region was biased to the C‐terminal region, which indicates the presence of a putative B‐cell epitope. The evaluation of the antigen specificity and determination of novel protective epitopes will provide valuable information for the further development and improvement of prophylactic HBV immunoglobulins.

The relationships between biophysical activity and the secondary structure of synthetic peptides from the pulmonary surfactant protein SP‐B

Synthetic pulmonary surfactants consisting of a mixture of phospholipids with synthetic peptides based on human and bovine surfactant‐associated protein SP‐B were prepared. These surfactants were analyzed for their biophysical activities by Wilhemly balance experiments and for their secondary structures by circular dichroism (CD) spectroscopy. Four synthetic peptides (SP‐1, SP‐2, SP‐3, and SP‐4) combined with the phospholipid mixture displayed significant surfactant properties. The CD spectra showed that the α‐helical propensities of the peptides in SDS micelles were related to their surfactant activities. These results suggested that the several truncated peptides originated from SP‐B protein, when appropriately recombined with phospholipids, could be used as an effective synthetic surfactant for clinical use.

Effects of treadmill exercise on hypoactivity of the hypothalamo-pituitary-adrenal axis induced by chronic administration of corticosterone in rats

The stress response alters behavior, autonomic function and secretion of multiple hormones, including CRF, ACTH, and glucocorticoid, through the HPA axis. Consecutive stress exposures lead to HPA axis dysregulation such as hyperactivity in Alzheimers disease and depression, and hypoactivity in post-traumatic stress disorder. In the present study, we established a model of hypoactivated HPA axis in rat through chronic administration of corticosterone (40mg/kg, s.c.) for 19 consecutive days. In this model, CRF mRNA expression in the hypothalamus and ACTH levels in serum were significantly decreased by chronic administration of corticosterone. In addition, the effect of treadmill exercise was investigated in our hypoactivated HPA axis rat model. Treadmill exercise recovered the dysregulated hypoactivity of the HPA axis induced by corticosterone administration for 19 days. The results of the present study suggest that treadmill exercise may aid recovery of hypoactivated HPA axis dysregulation in psychological diseases such as post-traumatic stress disorder.

Detection of surface asialoglycoprotein receptor expression in hepatic and extra-hepatic cells using a novel monoclonal antibody

The asialoglycoprotein receptor (ASGPR) is a heterodimeric membrane protein which is involved in the internalization of desialylated glycoproteins and also in the binding and uptake of various pathogenic viruses. To facilitate the analysis of ASGPR expression, we generated a monoclonal antibody, termed ASSA-1, that is specific to the ASGPR H1 subunit based on ELISA and Western blots analysis. ASSA-1 also reacted to surface-displayed ASGPR in live cells thus enabling analysis of ASGPR expression by immunofluorescence flow cytometry, which we used to analyze established human liver cell lines previously confirmed to be positive for ASGPR mRNA expression. In agreement with previous reports, surface ASGPR was also detected in extra-hepatic cells and, surprisingly, even in human T cell lines, which was then further confirmed in activated, but not in resting, primary human peripheral blood lymphocytes. These observations suggest that ASGPR has a broad pattern of expression that even extends into cells from the immune system, which biological meanings still have to be analyzed. We expect that monoclonal antibody ASSA-1 will serve as a new powerful tool in analyzing the biological role of ASGPR in hepatic and extra-hepatic cells.