Differentially expressed genes in silkworm cell cultures in response to infection by Wolbachia and Cardinium endosymbionts.

Wolbachia and Cardinium are bacterial endosymbionts that are widely distributed amongst arthropods. Both cause reproductive alterations, such as cytoplasmic incompatibility, parthenogenesis and feminization. Here we studied differentially expressed genes in Wolbachia- and Cardinium-infected Bm-aff3 silkworm cells using a silkworm microarray. Wolbachia infection did not alter gene expression or induce or suppress immune responses. In contrast, Cardinium infection induced many immune-related genes, including antimicrobial peptides, pattern recognition receptors and a serine protease. Host immune responses differed, possibly because of the different cell wall structures of Wolbachia and Cardinium because the former lacks genes encoding lipopolysaccharide components and two racemases for peptidoglycan formation. A few possibly non-immune-related genes were differentially expressed, but their involvement in host reproductive alteration was unclear.

Lipopolysaccharide elicits expression of immune-related genes in the silkworm, Bombyx mori.

Lipopolysaccharide (LPS), a major cell wall component of gram-negative bacteria, was found to be unable to activate immune-related genes in Drosophila melanogaster. In contrast, highly purified LPS elicited immune-related gene expression in the fat body of Bombyx mori. However, the level of activation by highly purified LPS was lower than crude LPS and peptidoglycan. Furthermore, synthetic lipid A also activated these genes, suggesting that B. mori possesses unknown signal pathways to activate immune-related genes by LPS. Up-regulation of antimicrobial peptide genes by highly purified LPS was not confirmed in the immune-responsive cell line, NIAS-Bm-aff3, suggesting that some factors necessary for signal transduction activated by LPS are deficient in this cell line.

Development and characterization of a continuous cell line, AFKM-On-H, from hemocytes of the European corn borer Ostrinia nubilalis (Hübner) (Lepidoptera, Pyralidae).

The corn borer, Ostrinia nubilalis, is a very important pest in different countries, and the in vitro system of the insect could be a useful tool for isolation and characterization of the pathogens and physiological responses of the insect. In this context, a cell line was derived from the hemocytes of the European corn borer and was named AFKM-On-H for, respectively, O. nubilalis, Armand Frappier, King Mongkut Institutes, and Hemocytes. This cell line was initiated and maintained in Ex-Cell 400 medium supplemented with 10% heat-inactivated fetal bovine serum. The cells, mostly spherical in shape, not firmly attached to the plastic culture flasks, were passaged up to 200 times by repeated gentle pipetting of the cells. The doubling times at the 80th and 125th passages at 28 degrees C and at the 122th and 169th passages at 25 degrees C were 40, 29, 35, and 34 h, respectively. The AFKM-On-H cell line was further characterized by the morphology, karyotype, random amplified polymorphic DNA analysis, and isozyme profiles. Susceptibility of the cell line to cytoplasmic polyhedrosis viruses (CPV) Euxoa scandens (EsCPV), Dendrolimus punctatus (DpCPV), and Choristoneura fumiferana (CfCPV); nuclear polyhedrosis viruses [Autographa californica (AcMNPV) wild type and recombinant, Antherea yammamai (AnyaNPV)]; and Chilo iridescent virus was demonstrated. Relative sensitivities of the cell line to Bacillus thuringiensis and Metarhizium anisopliae toxins and effects of the molting hormone 20-hydroxyecdysone on this new hemocyte cell line were characterized.

Interleukin-6 expression and gene polymorphism are associated with severity of periodontal disease in a sample of Brazilian individuals.

Interleukin (IL)-6 is an inflammatory mediator involved in bone resorption. G/C polymorphism at position -174 of the IL-6 gene has been reported to influence IL-6 expression, with the G allele associated with higher expression levels. The aims of this study were to investigate the expression of IL-6 as well as the incidence of IL-6 (-174) gene polymorphism and their correlation to the severity of periodontitis in Brazilians. Peripheral blood mononuclear cells were collected from 12 non-smoker individuals with periodontitis for evaluation of IL-6 expression using flow cytometry. We observed a positive correlation between the mean clinical attachment loss and intensity of expression of IL-6, in which the greater the attachment loss, the higher the expression of IL-6 (P=0 x 007, R2=0 x 52). Also, patients with severe periodontitis displayed a higher intensity of IL-6 expression compared to moderate periodontitis (P=0 x 04). To determine the occurrence of IL-6 gene polymorphism, DNA was obtained from oral swabs of 209 Brazilian individuals with and without periodontitis. Polymerase chain reaction, restriction endonuclease digestion and electrophoresis were performed, allowing for detection of the IL-6 (-174) polymorphism. We observed that non-smokers with moderate periodontitis (P=0 x 05) and control (P=0 x 04) groups displayed a higher incidence of the G genotype when compared to severe periodontitis. This suggests that the G genotype may represent a protective role in severity of periodontitis. Thus, the increased expression of IL-6 and IL-6 (-174) polymorphism are associated with periodontal disease severity in Brazilian individuals.

Establishment and characterization of cell-free translation/glycosylation in insect cell (Spodoptera frugiperda 21) extract prepared with high pressure treatment.

A coupled cell-free translation/glycosylation system, prepared from Spodoptera frugiperda insect cells, was established and optimized for protein production and glycosylation efficiency. Both translation and glycosylation were stimulated by addition of Mg2+, K+, ATP, GTP, creatine kinase and creatine phosphate, suggesting that glycoprotein productivity is largely determined by translation efficiency. However, high concentrations of creatine phosphate significantly inhibited translation. Spermidine stimulated both translation and glycosylation, but glycosylation required higher concentrations of spermidine than translation. Furthermore, extracts prepared at a nitrogen pressure of 10 kg/cm2 with the Mini-Bomb cell disruption chamber had the highest glycoprotein productivity; and extracts prepared at the higher nitrogen pressure of 15 kg/cm2 retained glycosylation ability. While extracts prepared with the Potter-Elvehjem homogenizer could mediate translation, no glycosylation was achieved. This indicated that the posttranslational machinery might survive disruption by high pressure, but not by physical shearing force. This insect cell-free system was able to synthesize approximately 25 microg of glycosylated gp120/ml of reaction mixture.

Peroral infectivity of non-occluded viruses of Bombyx mori nucleopolyhedrovirus and polyhedrin-negative recombinant baculoviruses to silkworm larvae is drastically enhanced when administered with Anomala cuprea entomopoxvirus spindles.

Non-occluded viruses (NOVs) of Bombyx mori nucleopolyhedrovirus (BmNPV) are poorly infectious to silkworm larvae when administered by peroral inoculation, although they are highly infectious when injected into the insect haemocoel. In the present study, it is demonstrated that NOVs of BmNPV became highly infectious even through peroral inoculation when administered with spindles (proteinaceous structures) of Anomala cuprea entomopoxvirus (AcEPV). Marked enhancement of peroral infectivity of NOVs by AcEPV spindles (nearly 1000-fold higher in the strongest case) was observed in all growth stages of silkworm larvae tested (2nd to 5th instar). Similarly, peroral infectivity of polyhedrin-negative recombinants of BmNPV, which do not produce polyhedra, was also enhanced remarkably by AcEPV spindles. In contrast, spheroids (proteinaceous structures containing AcEPV virions) did not enhance the peroral infectivity of either NOVs or the recombinant BmNPV in silkworm larvae.

Activation of background membrane conductance by the tyrosine kinase inhibitor tyrphostin A23 and its inactive analog tyrphostin A1 in guinea pig ventricular myocytes.

ABSTRACT-The effects of the tyrosine kinase (TK) inhibitor tyrphostin A23 and its inactive analog tyrphostin Al on background membrane conductance were investigated in guinea pig ventricular myocytes. TK-inhibiting A23 reversibly increased membrane conductance under conditions designed to minimize Na+, Ca2+, K+, and Na+-K+ pump currents. Similar stimulatory action was obtained with TK-inactive Al. The tyrphostin-induced current was inhibited by omitting external Na+ or Ca+, suppressed by chelating internal Ca2+, blocked by external Cd2+ and Ni2+, and insensitive to changes in internal Cl- concentration. We conclude that tyrphostins have a direct, TK-independent action that increases membrane conductance probably by stimulating Na+-Ca2+ exchange.

Differential level in co-down-modulation of CD4 and CXCR4 primed by HIV-1 gp120 in response to phorbol ester, PMA, among HIV-1 isolates.

HIV-1 enters cells through interacting with cell surface molecules such as CD4 and chemokine receptors. We generated recombinant soluble gp120s derived from T-cell line-tropic (T-tropic) and macrophage-tropic (M-tropic) HIV-1 strains using a baculovirus expression system and investigated the association of CD4-gp120 complex with the chemokine receptor and/or other surface molecule(s). For monitoring the co-down-modulations of the CD4-gp120 complex, a cytoplasmic domain deletion mutant (tailless CD4), which is not capable of undergoing down-modulation by itself in response to phorbol ester PMA, was used. Our studies revealed both cell-type and HIV-1 strain-specific differences. We found that T-tropic gp120s were capable of priming co-down-modulation with tailless CD4 by interacting with CXCR4, whereas M-tropic SF162 gp120 could not after PMA treatment even in the presence of CCR5. Among the T-tropic HIV-1 envelopes, IIIB gp120 was the most potent. Furthermore, the ability of gp120 to prime the PMA induced co-down-modulation of tailless CD4 appeared to be dependent on the concentration of the principal coreceptor CXCR4. Nevertheless, the observation that IIIB gp120 strongly primed tailless CD4 co-down-modulation on human osteosarcoma HOS cells that express undetectable levels of surface CXCR4 raised the possibility that membrane component(s) other than those recently identified can be involved in down-modulation of the CD4/gp120 complexes.

Sequence analysis of Pns11, a nonstructural protein of rice gall dwarf virus, and its expression and detection in infected rice plants and vector insects.

The nucleotide sequence of genome segment S11 of rice gall dwarf virus (RGDV), a member of Phytoreovirus, was determined. The segment encodes a putative protein of 40 kDa that exhibits approximately 37% homology at the amino acid level to the nonstructural proteins Pns10 of rice dwarf and wound tumor viruses, which are other members of Phytoreovirus. A band of a protein with an apparent molecular mass of 40 kDa was specifically detected in an analysis of cells transfected with S11 cDNA. An antiserum raised against this protein reacted with a protein of approximately 40kDa after fractionation by SDS-PAGE of materials prepared from infected plants and from viruliferous vector insects. However, the antiserum did not react with purified viral proteins. These results suggest that S11 encodes a nonstructural protein of RGDV. This protein was named Pns11.

Aspirin and salicylic acid do not inhibit methyl jasmonate-inducible expression of a gene for ornithine decarboxylase in tobacco BY-2 cells.

Similar to the prostanoid-mediated inflammatory response in mammals, jasmonate-mediated wound response in plant leaves is inhibited by salicylic acid (SA) or acetylsalicylate (aspirin). In tobacco BY-2 cells, expression of the gene for ornithine decarboxylase (ODC) involved in putrescine synthesis is rapidly inducible by methyl jasmonate (MeJA). A nuclear gene for ODC isolated from tobacco, gNtODC-1, was an intron-less gene and MeJA induced the expression of a GUS fusion gene with the gNtODC-1 promoter in transformed tobacco cells. Although SA alone did not induce the expression, 0.2 to 20 microM SA increased the MeJA-induced expression of the fusion gene to about two-fold. A similar increase was observed with aspirin but not with 3- or 4-hydroxybenzoic acids. SA at concentrations up to 200 microM did not inhibit the MeJA-induction of mRNAs for the GUS fusion gene and the endogenous gene for ODC.

A novel cell-free translation/glycosylation system prepared from insect cells.

A cell-free translation/glycosylation system derived from lepidopteran (Sf21) cells, which are widely used to express high yields of foreign active proteins that have post-translational modifications, was constructed. The insect cell extract was prepared using a Mini-Bomb cell disruption chamber by nitrogen pressure treatment, which stably retains translational and post-translational components. The gp120 mRNA was transcribed from the human immunodeficiency virus type-1 envelope glycoprotein gp120 gene with T7 RNA polymerase. When the gp120 mRNA was translated in the insect cell-free system, gp120 having a molecular mass of 100 kDa was detected by Western blot analysis. Synthesized gp120 and gp120 expressed in the intracellular fraction of recombinant-baculovirus-infected Sf21 cells had the same molecular mass, and they both had reduced mobility compared with gp120 secreted by recombinant baculovirus-infected Sf21 cells. In contrast, the 56-kDa gp120 protein, which corresponds to the polypeptide backbone of gp120, was synthesized in wheat germ and rabbit reticulocyte systems. The molecular mass of synthesized gp120 decreased from 100 kDa to 61 kDa after endoglycosidase H treatment, indicating that synthesized gp120 had been glycosylated with N-linked oligosaccharides. Furthermore, glycosylated gp120 was bound to human CD4 molecules expressed on the surface of quail cells. These results revealed that the insect cell-free system can synthesize gp120 that is folded in the proper conformation to provide a CD4-binding domain.

Expression patterns of two genes for the delta-subunit of mitochondrial F1-ATP synthase from sweet potato in transgenic tobacco plants and cells.

Two nuclear genes, F1 delta-1 and F1 delta-2, coding for the delta-subunit of mitochondrial F1-ATP synthase, which corresponds to oligomycin-sensitivity conferring protein in animal and yeast mitochondria, were isolated from sweet potato. The gene for the delta-subunit was composed of 6 exons and these two genes shared high sequence similarities to each other not only in exons but also in introns and in the 5'-upstream regions. However, the 5'-upstream regions of F1 delta-1 and F1 delta-2 were distinguishable by the presence of novel sequences, designated Ins-1 and Ins-2, respectively. Ins-1 and Ins-2 contained a terminal direct repeat of 10 bp and 12 bp, respectively, and various forms of repeat sequences. The promoter fusion of both F1 delta-1 and F1 delta-2 with the GUS coding sequence gave expression of GUS activity in transformed tobacco BY-2 cells, although the levels of GUS activity and the patterns of expression during the growth of cells were different between the two. In transgenic tobacco plants, the two fusion genes showed similar levels of expression in leaves and stems, while F1 delta-2:GUS gave significantly higher levels of expression in roots than F1 delta-1:GUS. Deletion of Ins-1 from the 5'-upstream region of F1 delta-1:GUS did not affect the expression of the fusion gene in various organs of transgenic plants. However, it caused significant enhancement of expression in transformed tobacco BY-2 cells.

Mechanisms of cation permeation in cardiac sodium channel: description by dynamic pore model.

The selective permeability to monovalent metal cations, as well as the relationship between cation permeation and gating kinetics, was investigated for native tetrodotoxin-insensitive Na-channels in guinea pig ventricular myocytes using the whole-cell patch clamp technique. By the measurement of inward unidirectional currents and biionic reversal potentials, we demonstrate that the cardiac Na-channel is substantially permeable to all of the group Ia and IIIa cations tested, with the selectivity sequence Na(+) >/= Li(+) > Tl(+) > K(+) > Rb(+) > Cs(+). Current kinetics was little affected by the permeant cation species and concentrations tested (

Differential induction by methyl jasmonate of genes encoding ornithine decarboxylase and other enzymes involved in nicotine biosynthesis in tobacco cell cultures.

A cDNA of tobacco BY-2 cells corresponding to an mRNA species which was rapidly induced by methyl jasmonate (MeJA) in the presence of cycloheximide (CHX) was found to encode ornithine decarboxylase (ODC). Another cDNA from a MeJA-inducible mRNA encoded S-adenosylmethionine synthase (SAMS). Although these enzymes could be involved in the biosynthesis of polyamines, the level of putrescine, a reaction product of ODC, increased slowly and while the levels of spermidine and spermine did not change following treatment of cells with MeJA. However, N-methylputrescine, which is a precursor of pyrrolidine ring of nicotine, started to increase shortly after MeJA-treatment of cells and the production of nicotine occured thereafter. The levels of mRNA for arginine decarboxylase (ADC), an alternative enzyme for putrescine synthesis, and that for S-adenosylmethionine decarboxylase (SAMDC), required for polyamine synthesis, were not affected by MeJA. In addition to mRNAs for ODC and SAMS, mRNA for putrescine N-methyltransferase (PMT) was also induced by MeJA. Unlike the MeJA-induction of ODC mRNA, MeJA-induction of SAMS and PMT mRNAs were blocked by CHX. The level of ODC mRNA declined after 1 to 4 h following MeJA treatment, while the levels of mRNAs for SAMS and PMT continued to increase. Auxin significantly reduced the MeJA-inducible accumulation of mRNAs for ODC, SAMS and PMT. These results indicate that MeJA sequentially induces expression of a series of genes involved in nicotine biosynthesis by multiple regulatory mechanisms.

Effect of sulfhydryl oxidoreduction on permeability of cardiac tetrodotoxin-insensitive sodium channel.

Effects of sulfhydryl oxidizing and reducing agents on permeability of the tetrodotoxin (TTX)-insensitive Na-channel were investigated in guinea-pig ventricular myocytes using the whole-cell patch-clamp technique. Mercury chloride (HgCl2) at 1-100 microM irreversibly blocked Na+ currents with no significant changes in the gating kinetics. In contrast, the hydrophilic sulfhydryl oxidizing agent, thimerosal at 50-100 microM little affected Na+ permeation through the Na-channel. The Hg2+-induced block of Na+ current could be readily reversed by 1,4-dithiothreitol (DTT), an agent that reduces disulfide bonds. These results indicate that the formation of sulfur-Hg-sulfur bridge is essential for Hg2+ block. Pretreatment with DTT prevented the Hg2+ block of Na+ current, whereas Zn2+ and Cd2+ retained their abilities to block Na+ current after DTT treatment. An application of Zn2+ or Cd2+ resulted in the restoration of Hg2+ sensitivity of the DTT-treated channel. A conformational model for the Na-channel with multiple free sulfhydryl groups and native disulfide bonds could account for our experimental data regarding the effects of sulfhydryl modifying agents on the channel permeability. We conclude that the cardiac TTX-insensitive Na-channel contains functionally important free sulfhydryl groups and disulfide bonds which are accessible from the extracellular side by an aqueous pathway. These sulfhydryls would be capable of modulating the Na-channel permeability by affecting the conformation of channel pore region.

Brain potentials associated with eye fixations during visual tasks under different lighting systems.

The variations of eye fixation related potentials (EFRPs) were examined in two tasks under three lighting conditions for assessment of lighting environments. Sixteen subjects participated in two tasks; a difficult and an easy reading task under three lighting conditions: Spot light (S), General light (G) and Mixed light (M). EEG (Oz) and EOG were recorded. EEG epochs time-locked to onset of eye fixations were collected at random and averaged separately in two arrays to obtain a pair of EFRPs. Two wave forms under the S were similar, although those under the G showed the disparity, the largest disparity being in the easy task under the G. Under the S, wave forms of EFRPs were stable in the difficult task. The amplitude changed with the task load. The results suggested that EFRPs might be an index of the work load under lighting conditions.

An mRNA of tobacco cell, which is rapidly inducible by methyl jasmonate in the presence of cycloheximide, codes for a putative glycosyltransferase.

Two-dimensional gel electrophoretic display of polypeptides labeled in vivo and those synthesized in vitro from poly(A)(+)-RNA indicated that treatment of cultured cells of tobacco (Nicotiana tabacum) BY-2 with methyl jasmonate (MeJA) induces accumulation of a limited number of specific mRNAs within a few hours. The MeJA-induction of most of these mRNAs was inhibited by cycloheximide (CHX). Six MeJA-inducible cDNAs identified by differential screening were classified into three groups based on the sensitivity of their induction to CHX. Induction of group I mRNAs by MeJA occurred earlier than the induction of other mRNAs and it was not inhibited by CHX. The induction of group II mRNAs by MeJA was blocked by CHX, while group III mRNAs were induced by CHX alone. One group I cDNA was found to encode a putative protein, JIGT, homologous to UDP-sugar glycosyltransferases previously characterized from several plant species. JIGT was structurally different from a putative glycosyltransferase that is rapidly inducible by salycylic acid (SA) in BY-2 cells. JIGT mRNA was not induced by SA. In addition to MeJA, as little as 10(-9) M coronatine induced JIGT mRNA. A sequence highly homologous to JIGT is present as a single copy in the genomes of Nicotiana sylvestris and N. tomentosiformis. The MeJA-inducible production of JIGT may be involved in sugar-conjugation of an unknown substrate in a defensive response and expression of the gene for JIGT in BY-2 cells might serve as a good model system for disecting molecular events occurring in JA-inducible gene expression.

A major jasmonate-inducible protein of sweet potato, ipomoelin, is an ABA-independent wound-inducible protein.

Treatment of sweet potato plants cultured in vitro with a vapor of methyl jasmonate (MeJA) induced an accumulation in leaves of a large amount of protein with an apparent molecular mass of 18 kDa. This protein, designated ipomoelin, was purified, and the amino acid sequences of proteolytic fragments were determined. Screening a cDNA library of MeJA-treated leaves by oligonucleotide probes designed from the peptide sequences identified a clone that could code for a polypeptide with 154 amino acids. The deduced amino acid sequence of ipomoelin showed an overall amino acid identity of 25% with the salt-inducible SalT protein of rice. In addition, the C-terminal 70 amino acid sequence of ipomoelin showed about 50% identity with the C-terminal amino acid sequences of seed lectins from Moraceae. The gene for ipomoelin was present in a few copies in the genome of sweet potato. The mRNA for ipomoelin was detected in leaves and petioles, but not in stems and tuberous roots, of sweet potato plants grown in the field. Mechanical wounding of leaves induced ipomoelin mRNA both locally and systemically, while treatment of leaves with ABA, salt, or a high level of sucrose did not induce ipomoelin mRNA. By contrast, ABA-inducible mRNA for sporamin was not induced by MeJA. These results suggest that ipomoelin is involved in defensive reactions of leaves in response to wounding and that JA-mediated wound-induction of ipomoelin occurs independently of ABA.

Diverse inotropic effects of 5-hydroxytryptamine in heart muscles of various mammalian species.

The inotropic effects of 5-hydroxytryptamine (5-HT) on mammalian heart muscles were investigated. 5-HT (10(-8)-10(-3)M) produced increases in the contractile tension of atrial and ventricular muscles isolated from guinea pigs, Japanese monkeys, and humans, but not in rat heart preparations. The maximum percent increase of contraction was largest in guinea pig ventricular muscles (142.0 percent), followed by monkey atrium (86.3 percent), human atrium (71.7 percent), guinea pig atrium (48.7 percent), and monkey ventricle (30.1 percent). The sensitivity to 5-HT, measured as the negative logarithm of the half-maximal inotropic molar contractions of 5-HT, i.e., -logEC(50), was highest in the human atrium (6.65 +/- 0.20), followed by guinea pig atrium (5.53 +/- 0.36), monkey ventricle (4.83 +/- 0.28), guinea pig ventricle (4.56 +/- 0.11), and monkey atrium (4.46 +/- 0.16). The inotropic effects of 5-HT seen in the atrial and ventricular muscles of guinea pigs were abolished in the presence of the beta-receptor blocker, pindolol (8 mu M), while these effects in human atrial muscles and monkey atrial and ventricular muscles were abolished only in the presence of both pindolol (8 mu M) and of prazosin (1 mu M), an alpha(1)-receptor blocker. 5-HT increased the V(max) of the slow response recorded from guinea pig ventricular muscles exposed to high K+ (27 mM) media, whereas this agent did not alter the calcium current of isolated guinea pig ventricular myocytes devoid of sympathetic nerve terminals. In reserpinized guinea pig hearts, 5-HT exerted no inotropic effect on ventricular muscle, yet it had an inotropic effect in the atrial muscle, although the latter effect was considerably depressed, compared to that seen in non-reserpinized atrial muscles. We conclude that the positive inotropic effects of 5-HT observed in the ventricular muscle of the guinea pig and in the atrial and ventricular muscles of the Japanese monkey can be attributed to the release of noradrenaline from sympathetic nerve terminals (indirect effect). In contrast, in human atrial muscles, the positive inotropic effect of 5-HT was apparently the result of stimulation of a specific membrane receptor for 5-HT (direct effect). In guinea pig atrial muscles, both direct and indirect effects of 5-HT were involved in the positive inotropism. An explanation for the lack of sensitivity of rat atrial and ventricular muscles to 5-HT awaits further studies.