The effect of ganglioside GQ1b on the NMDA receptor signaling pathway in H19-7 cells and rat hippocampus.

Gangliosides, sialic acid-containing glycosphingolipids, are related to various synaptic functions in the rat brain. Previously, we investigated the behavioral effects of the ganglioside GQ1b on learning and memory using the Y-maze and Morris water maze test. GQ1b-treated rats showed highly increased memory performance on the Y-maze and the Morris water maze test. In this study, we determined the role of GQ1b on the activation of the N-methyl-d-aspartate (NMDA) receptor signaling pathway in H19-7 rat hippocampal cells and the hippocampus of rats. After 12 h of treatment with GQ1b, the expression levels of NMDA receptor subunit 2A and 2B were increased in H19-7 cells and the hippocampus of rats. In addition, treatment of GQ1b increased the tyrosine phosphorylation of NR2B that may enhance NMDA receptor synaptic activation and enhancement of NMDA receptors. Also, following GQ1b treatment, the phosphorylation of extracellular signal-regulated kinases (ERK1/2) and protein kinase A, a cAMP activated protein kinase (PKA) increased in H19-7 cells and the hippocampus of rats. These increases resulted in an increase in the phosphorylation of cAMP response element binding protein (CREB). These results suggest that GQ1b might facilitate the activation of the NMDA receptor signaling pathway in the hippocampus of rats, an effect which is dependent on ERK1/2, PKA and CREB phosphorylation. Also, these data support our previous result that GQ1b improves the learning and memory of rats.

A novel chimeric promoter that is highly responsive to hypoxia and metals.

To develop a potent hypoxia-inducible promoter, we evaluated the usefulness of chimeric combinations of the (Egr-1)-binding site (EBS) from the Egr-1 gene, the metal-response element (MRE) from the metallothionein gene, and the hypoxia-response element (HRE) from the phosphoglycerate kinase 1 gene. In transient transfection assays, combining three copies of HRE (3 x HRE) with either EBS or MRE significantly increased hypoxia responsiveness. When a three-enhancer combination was tested, the EBS-MRE-3 x HRE (E-M-H) gave a hypoxia induction ratio of 69. The expression induced from E-M-H-pGL3 was 2.4-fold higher than that induced from H-pGL3 and even surpassed the expression from a human cytomegalovirus promoter-driven vector. The high inducibility of E-M-H was confirmed by validation studies in different cells and by expressing other cDNAs. Gel shift assays together with functional overexpression studies suggested that increased levels of hypoxia-inducible factor 1alpha, metal transcription factor-1 and Egr-1 may be associated with the high inducibility of the E-M-H chimeric promoter. E-M-H was also induced by hypoxia mimetics such as Co2+ and deferoxamine (DFX) and by hydrogen peroxide. Gene expression from the E-M-H was reversible as shown by the reduced expression of the transgene upon removal of inducers such as hypoxia and DFX. In vivo evaluation of the E-M-H in ischemic muscle revealed that erythropoietin secretion and luciferase and LacZ expression were significantly higher in the E-M-H group than in a control or H group. With its high induction capacity and versatile means of modulation, this novel chimeric promoter should find wide application in the treatment of ischemic diseases and cancer.

HLA-DR expression in B-lymphocytes in vitro is not suppressed by the absence of exogenous antigens.

The proper loading of exogenous peptide antigens affects the transport and cell surface expression of MHC class II molecules. In the present study, the goal was to determine to what extent this step determines the cell surface expression level of MHC class II molecules, such as the HLA-DR. EBV-transformed B-cells, were cultured either in a serum- and protein-free medium, or in a medium that contained different concentrations of exogenous antigens. Using HLA-DR-specific antibodies, the induction of the MHC class II expression was observed in cells that were cultured under serum-and protein-free conditions, when compared to those cultured with exogenous protein antigens. This upregulation was completely suppressed to the normal level by the addition of a high concentration of hen egg lysozyme to the serum- and protein-free medium. This indicates that exogenous proteins regulate the HLA-DR expression. To further examine whether this modulation is controlled at the transcription level, the expression of the HLA-DR beta-chain mRNA was analyzed by reverse transcription-PCR and Northern blots. The same levels of HLA-DRB mRNA were detectable in both culture conditions, indicating that the present observation is dependent on some regulatory mechanisms at the post-transcriptional level. This might include a different pathway for trafficking of HLA-DR molecules to the cell surface, since peptide-binding assays revealed that a high proportion of cell surface HLA-DR molecules under the serum- and protein-free condition were transported to the cell surface without associated peptide antigens.

Recombinant expression of biologically active rat leptin in Escherichia coli.

Leptin is a 16-kDa nonglycosylated hormone that is produced in mature adipocytes and which acts primarily in the hypothalamus to reduce food intake and body weight. While the rat is a representative laboratory animal model in obesity research, so far recombinant rat leptin was not available. In the present study, rat leptin was recombinantly expressed in Escherichia coli and purified in a bioactive form to provide a further tool for the analysis of leptin functions in rats. Leptin cDNA was cloned by RT-PCR from total RNA of SD rat adipocytes, and overexpression was achieved by subcloning the leptin cDNA into the pET-29a vector, which enabled the recombinant expression of rat leptin as an S-peptide-tagged fusion protein. Since the fusion proteins were expressed in inclusion bodies, after purification of the insoluble fraction, leptin proteins were refolded by sequential dialysis into physiological buffers. The biological activity of this recombinant protein was confirmed in proliferation assays using leptin-sensitive rat insulinoma cells as well as a newly developed leptin-sensitive luciferase assay system. The specific binding of the S-tagged leptin to leptin-receptor-expressing cells was further shown by flow cytometry using fluorescence-conjugated S-proteins.

Translocation and accumulation of exogeneous hepatitis B virus preS surface proteins in the cell nucleus.

Recurrent reports about protease-sensitive sites in the junction of the preS and S region of the hepatitis B virus large surface protein have raised the question about a possible biological role of S protein-depleted, independent preS protein fragments in the virus life cycle. In the present study, this question was addressed by exogenous introduction of fluorescence-labeled recombinant preS proteins into permeabilized HepG2 cells. While maltose-binding proteins (MBP) were evenly distributed throughout the cytoplasm, MBP-preS fusion proteins selectively accumulated in the nucleus. Using truncated preS proteins, the effective domain for this nuclear accumulation was localized around the preS2 region. The mode of this action differs from conventional nuclear translocation mechanism in its energy- and mediator-independency and in that it is not saturated regardless of the increase of preS protein concentration. The biological meaning of this phenomenon has to be further studied. However, in regard to hepatitis B virus infection, this observation might provide a clue for unveiling the still poorly characterized events after initial internalization of the virus, which might make use of the nuclear translocation effect of the preS2 region to facilitate the infection.

Detection of cellular receptors specific for the hepatitis B virus preS surface protein on cell lines of extrahepatic origin.

Hepatitis B virus infection is primarily mediated by the interaction of the preS region of the viral envelope protein with its still unknown cellular receptor. Using recombinantly expressed preS proteins, the distribution of preS-binding receptors on cell lines from extrahepatic origins was determined by immunofluorescence and flow cytometry. In contrast to human liver cell lines, most cell lines from extrahepatic origins did not bind preS proteins. Nevertheless, exceptions were found in the bone marrow-derived cell line, KG-1, and the osteogenic sarcoma cell line SaOS-2, as well as in the previously reported EBV-transformed B-cell line, Wa. To determine the biochemical nature of these receptors, Wa-cells were cell surface biotinylated and the preS-binding receptors were isolated by immunoprecipitation. A specific band with a molecular weight of approximately 30 kDa was identified in a SDS-polyacrylamide gel, which further characterization is expected to provide clues regarding the infection mechanism of HBV in hepatic- and extra-hepatic cells.

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.

Downregulation of MHC class II expression by oxidant-induced apoptosis in EBV-transformed B-cells.

The expression of MHC class II molecules is actively regulated upon various cellular stimuli. Since apoptosis is an inducible cellular process, it was asked whether cells undergoing apoptosis would also modulate their expression of class II molecules. Using an EBV-transformed B-cell line, the cell surface expression of HLA-DR molecules was analyzed by fluorescence-activated flow cytometry on normal and oxidant-treated apoptotic cells. A rapid and continuous decrease in HLA-DR expression was observed in apoptotic cells. RNA analysis and semiquantitative RT-PCR of cytoplasmic beta-actin mRNA showed that apoptotic cells contain partially degraded RNA and much lower amounts of beta-actin mRNA. Nevertheless, when compared after normalization of intact mRNA amounts, the HLA-DRB mRNA signals were of similar strength in normal and apoptotic cells as determined by semiquantitative RT-PCR. Thus, the decrease in the number of class II molecules during apoptosis underlies no specific program for downregulation of HLA-DRB mRNA transcription but is due to a nonspecific degradation of RNA molecules accompanied by cell death.

Isolation and characterization of a novel antifungal peptide from Aspergillus niger.

A novel antifungal peptide (termed as Anafp) was isolated from the culture supernatant of the filamentous fungi, Aspergillus niger. The whole amino acid sequence of Anafp was determined and the peptide was found to be composed of a single polypeptide chain with 58 amino acids including six cysteine residues. The peptide shows some degree of sequence homology to a cysteine-rich antifungal peptides reported from the seeds of Sinapis alba and Arabidopsis thaliana or the extracellular media of Aspergillus giganteus and Penicillium chrysogenumsome. Cysteine-spacing pattern of Anafp was similar to that of the antifungal peptide from Penicillium chrysogenum. The Anafp exhibited potent growth inhibitory activities against yeast strains as well as filamentous fungi at a range from 4 to 15 microM. In contrast, Anafp did not show antibacterial activity against Escherichia coli and Bacillus subtilis even at 50 microM.

Effects of tryptophan residues of porcine myeloid antibacterial peptide PMAP-23 on antibiotic activity.

PMAP-23 is a 23-residue antimicrobial peptide from porcine myeloid cells. In order to determine the effects of two Trp residues in positions 7 and 21 of PMAP-23 on antibacterial activity and phospholipid vesicle interacting property, two analogues in which Ala is substituted for Trp residue in position 7 or 21 were synthesized. A(21)-PMAP-23 exhibited reduced antibacterial activity and phospholipid vesicle disrupting activity when compared to those of PMAP-23 and A(7)-PMAP-23. PMAP-23 readily interacted with model lipid membrane and induced membrane destabilization. Therefore antibacterial activity induced by PMAP-23 is due to the interaction of cell membrane with peptide followed by membrane perturbation. A significant structural change on the SDS micelle was not found by Ala substitution of the Trp residue of PMAP-23. Also, there is a good correlation between hydrophobic interaction on RP-HPLC, expressed as retention time on RP-HPLC, and antibacterial activity. The vesicle titration experiment indicated that Trp residues located at near C-terminus are accessible to hydrophobic tail of phospholipid vesicle. This result suggests that the C-terminal end of PMAP-23 penetrates into the lipid bilayer in the course of the interaction with phospholipid membranes and is important for its antibacterial activity.

NMR structural characterization of cecropin A(1-8) - magainin 2(1-12) and cecropin A (1-8) - melittin (1-12) hybrid peptides.

In order to elucidate the structure-antibiotic activity relationships of the peptides, the three-dimensional structures of two hybrid peptides, CA(1-8) - MA(1-12) and CA(1-8) - ME(1-12) in trifluoroethanol-containing aqueous solution were investigated by NMR spectroscopy. Both CA(1-8) - MA(1-12) and CA(1-8) - ME(1-12) have strong antibacterial activity but only CA(1-8) - ME(1-12) has hemolytic activity against human erythrocytes. CA(1-8) - MA(1-12) has a hydrophobic 310-helix of only two turns combined with one short helix in the N-terminus with a flexible hinge section in between. CA(1-8) - MA(1-12) has a severely bent structure in the middle of the peptide. These structural features as well as the low hydrophobicity of CA(1-8) - MA(1-12) seem to be crucial for the selective lysis against the membrane of prokaryotic cells. CA(1-8) - ME(1-12) has an alpha-helical structure of about three turns in the melittin domain and a flexible structure with one turn in the cecropin domain connected with a flexible hinge section in between, and these might be the structural features required for membrane disruption against prokaryotic and eukaryotic cells. The central hinge region (Gly9-Ile10-Gly11) in an amphipathic antibacterial peptide is considered to play an important role in providing the conformational flexibility required for ion channel formation of the C-terminal hydrophobic alpha-helix on cell membrane.

Detection of the asialoglycoprotein receptor on cell lines of extrahepatic origin.

The asialoglycoprotein receptor (ASGPR) is the first lectin discovered in mammals. Despite its significant biological role in binding and internalization of desialyated glycoproteins, at least in the human, little information is available regarding its tissue distribution outside of the liver. In the present study, antibodies were raised against the H1 major subunit of the human ASGPR using synthetic peptide antigens, and their binding specificity confirmed by enzyme linked immunosorbent assay. Cell surface analysis by fluorescence activated flow cytometry on various human tissue cell lines confirmed the liver parenchymal cells as the major expression site of ASGPR. Nonetheless, ASGPR was also detectable on some extrahepatic cells such as the Jurkat T-cell line. The determination of extrahepatic expression of ASGPR will have consequences in analyzing the biological role of this receptor complex as well as having implications in designing ASGPR mediated drug- or gene-delivery strategies.

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 alpha-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.

Structure-antitumor and hemolytic activity relationships of synthetic peptides derived from cecropin A-magainin 2 and cecropin A-melittin hybrid peptides.

The hybrid peptide (CA-ME) derived from cecropin A(1-8) and melittin (1-12) has potent antibacterial and antimalarial activities. Because the N-terminal sequence 1-12 of magainin 2 is similar to melittin(1-12), CA-MA with CA(1-8) and MA(1-12) and their analogues were designed and synthesized. Antitumor activities of these peptides were evaluated using three small cell lung cancer cell lines. Greater antitumor activity was observed when the residues 16, 18 and 19 of the peptide were hydrophobic (Leu or Val), basic (Lys) and basic (Lys), respectively. The IC50 values of the peptides with the residues were 2 to 4 microM. Residue 12 was related to hemolytic activity rather than antitumor activity. Increase in amphipathicity of P4 enhanced hemolytic activity without significant change in antitumor activity. The alpha-helicity of the peptides in a 30 mM sodium dodecyl sulfate solution was more closely correlated to hemolytic activity than antitumor activity.

Generation and characterization of a novel fusion partner cell line for the production of human macrophage hybridoma.

Macrophages are important constituents of the immune system by exerting phagocytosis on invading pathogens as well as secreting various immunoregulatory factors. Generation of human macrophage hybridoma has not been possible so far due to the lack of an appropriate fusion partner cell line. In the present study, an 8'-azaguanine resistant cell line, termed HL-60R, was established by drug selection of the promyelocytic cell line HL-60. This novel cell line showed resistance to high concentrations of 8'-azaguanine and was sensitive to aminopterin. These characteristics make it suitable for serving as a potential fusion partner cell line in the development of macrophage hybridoma. Cell-surface analysis by FACS revealed that HL-60R cells per se do not express MHC-class II molecules or the macrophage marker, CD11b. PEG-mediated fusion of HL-60R was performed with PBMC-derived human macrophages. Fluorescence labelling of ex vivo isolated macrophages prior to fusion and subsequent FACS analysis showed that PEG-4000 is a more effective fusion agent than PEG-1500. The generation of this novel fusion partner cell line opens the possibility for development of human macrophage hybridoma or other cell lines from myelocytic origin. Such hybridoma clones will not only enable a more convenient study of these cell but will also provide an excellent host site for the proper production and expression of various recombinant proteins from myelocytic origin in vitro.

Binding kinetics of monoclonal antibody using antigen-beta-galactosidase hybrid protein: application to measurement of peptide antigenicity.

A simple method for determination of binding kinetics of a solid-phase antibody using antigen-beta-galactosidase hybrid protein was evaluated. To minimize conformational change of the antigen binding site of the antibody when directly binding to a microtiter plate, the microtiter plate was precoated with protein A. The binding and free antigen concentrations were directly obtained from the beta-galactosidase activity. This method can be used for analyses of the equilibrium dissociation constant (KD), and the association (Kass) and dissociation (Kdiss) rate constants. Peptide antigenicity was also analyzed by competitive ELISA using this method. Since both antigen-beta-galactosidase and the peptide used are localized in the fluid-phase, the proper affinity constant (KA) of the peptide can be estimated from the KD value of the antigen-beta-galactosidase-antibody interaction, and from the IC50 value of the peptide.

Structure-antigenicity relationship of peptides from the pre-S2 region of the hepatitis B virus surface antigen.

Peptide antigenicity against the pre-S2 region of the hepatitis B virus surface antigen was studied using a pre-S2 specific anti-hepatitis B virus mouse monoclonal antibody (H8 mAb) and synthetic peptides by competitive ELISA. The mAb showed preferences for long peptides with the sequence 120/123-145, though the mAb binding region was located in the sequence 130-145 from the analysis of a conjugation study. The N-terminal residues 120/123-129 play an important role for the maintenance of the highly antigenic structure of the B cell epitope. Among these, the N-terminal hydrophilic residues 124-126 and hydrophobic residue 127 were important, whereas residues 120-122 did not affect antigenicity. Residues 131 and 141 appeared to be critical for the mAb binding. The relationship between peptide structure and antigenicity was also investigated by probing the secondary structure of the peptides by circular dichroism. Highly antigenic peptides elicited more ordered structure in 20% trifluoroethanol than less antigenic peptides. The results suggested that peptide antigenicities against H8 mAb are closely related to the B-cell epitope conformations of peptides.

Aromatase, 5-alpha-reductase, and androgen receptor levels in the fetal monkey brain during early development.

Aromatase, 5 alpha-reductase and cytosolic androgen receptor levels were measured in the medial basal hypothalamus (MHB), amygdala (AMG), cerebellum and cerebral cortex of male and female fetal rhesus monkeys on day 70 of gestation. Higher aromatase activities were noted in the MBH and AMG of male than female fetuses. In contrast, no sex differences were found for 5 alpha-reductase and androgen receptor levels. These data suggest that at this early stage of development, differentiation of the MBH and AMG of the male fetus may be more susceptible to androgen modification, by way of aromatization to estrogens, than corresponding areas in the female fetus. Moreover, based upon a comparison of the current data to that published previously for later stages of development, it is suggested that the sex differences in aromatase activity are not the result of androgen stimulation.

Androgen receptors are differentially distributed between right and left cerebral hemispheres of the fetal male rhesus monkey.

In humans there are apparent sex differences in verbal and spatial abilities as well as several cortical pathologies. These differences may arise as the result of prenatal androgen exposure and its effect on the development of the cerebral cortex. With this in mind, we have examined androgen receptor (AR), aromatase (AROM) and 5 alpha-reductase (5 alpha R) levels in the cerebral cortex of Day 70 male and female fetal rhesus monkeys (Macaca mulatta). Receptor and enzyme levels were evaluated in both right (Rt) and left (Lft) temporal (TMP) and frontal (FR) lobes of the cerebral cortex. AR levels in FR-Rt of male subjects were higher than levels in FR-Lft (for each and every subject, P less than 0.05), while in females, there was no consistent pattern in the distribution of the receptor between the two sides of FR. In contrast, AR values in TMP-Lft of male subjects were consistently higher than in TMP-Rt (P less than 0.05). As with the FR, females exhibited no consistent pattern in the distribution of AR between the two TMP sides. AROM and 5 alpha R levels were similar, regardless of sex, between both sides of the two cortical lobes indicating that the AR distribution pattern is not a general biochemical phenomenon. The differential cortical distribution of AR in fetal males versus females lends support to the hypothesis that prenatal androgens from the fetal testes may effect the differentiation of sexually dimorphic, side-specific cortical activity.

Estrogen receptors in the rhesus monkey brain during fetal development.

Estrogen receptor (ER) levels were measured in brain tissue cytosol from fetal male and female rhesus monkeys at Days 70, 100 and 160 postconception. The brain regions which were examined included medial basal hypothalamus (MBH), amygdala (AMG), cerebral cortex (CTX) and cerebellum (CB). For comparison, brain tissues were also obtained from an adult female, and muscle (MUS) and genital tract (GEN, ovaries + uterus) ER values were measured in several Day 70 fetuses. Tissues were dissected and homogenized as previously described. Cytosol was passed through a microcolumn of Lipidex 1000 to remove interfering lipids and incubated with [3H]Moxestrol (4 nM) in the presence or absence of 500 nM Moxestrol. Incubations were carried out for 24 h at 4 degrees C, and free and bound ligand separated by Sephadex LH-20 gel filtration. In one case (Day 160 male fetus), saturation analysis yielded an estimate of apparent Kd of 0.46 x 10(-9) M and indicated that maximal specific binding was achieved at a ligand concentration of 1-2 nM. There was no sex difference at any stage of development (ANOVA). A significant age effect (P less than 0.002) was noted for the MBH and CB but not for any of the other tissues examined. In the MBH the significance of this effect was due to a progressive increase in ER levels with fetal age and into adulthood. In contrast, CB levels exhibited a progressive decline with age. These studies revealed that the ER is present during brain development. Thus any estrogens derived from the aromatization of circulating fetal androgens could potentially exert an influence upon brain development.(ABSTRACT TRUNCATED AT 250 WORDS)