Rosa pinna (Clerodendrum philippinum Schauer), is a shrub with attractive and fragrant flowers. However, the tall and spread architecture and high leaf to flower ratio hinder its use as an ornamental plant. Hence, changing the level of acceptance of C. philippinum by incorporating a dwarfing gene to the plant is considered useful. A study was conducted to find a suitable regeneration medium and ultimately to modify the plant architecture through transformation of the gibberellic acid inhibitor (gai) mutant dwarfing gene by particle bombardment. Effects of different concentrations of thidiazuron (TDZ:Nphenyl N’ 1,2,3-thidiazol-5-yl urea) on callus initiation and regeneration of shoots and the effects of micro career flying distance in gene transformation of C. philippinum were investigated. The regenerated shoots were first cultured on hormone free MS medium and transferred to a MS medium with 0.5 mg/L in dole butyric acid for rooting after four weeks. The shortest duration for callus initiation, the largest callus volume (28 days after establishment) and the highest number of shoots (75 days after establishment) were observed in 1.5 mg/L TDZ in the MS medium. Results of transferring the gai mutant gene to in vitro leaves of C. philippinum revealed that both the shortest time duration and the highest plant regeneration were at 9 cm micro-carrier flying distance. Leaf tissues of regenerated plants, which carry the gai mutant gene, positively responded for the beta-glucuronidase (GUS) assay. The regenerated shoots were rooted and successfully acclimatized.Tropical Agricultural Research Vol. 25 (1): 27-37 (2013)

Shen, J.; Perera, P.-Y.; Zhang, G.; Voqel, S. N.; Morrison, D. C.; Qureshi, N. Author Information

Qureshi, N.; Perera, P.-Y.; Shen, J.; Zhang, G.; Lenschat, A.; Vogel, S. N.; Morrison, D. C. Author Information

We have developed a novel LPS probe using a highly purified and homogenous preparation of [3H] Escherichia coli LPS from the deep rough mutant, which contains a covalently linked, photoactivable 4-p-(azidosalicylamido)-butylamine group. This cross-linker was used to identify the LPS-binding proteins in membranes of the murine-macrophage-like cell line RAW 264.7. The α-subunit (PSMA1 C2, 29.5 kDa) and the β-subunit (PSMB4 N3, 24.36 kDa) of the 20S proteasome complex were identified as LPS-binding proteins. This is the first report demonstrating LPS binding to enzymes such as the proteasome subunits. Functionally, LPS enhanced the chymotrypsin-like activity of the proteasome to degrade synthetic peptides in vitro and, conversely, the proteasome inhibitor lactacystin completely blocked the LPS-induced proteasome’s chymotrypsin activity as well as macrophage TNF-α secretion and the expression of multiple inflammatory mediator genes. Lactacystin also completely blocked the LPS-induced expression of Toll-like receptor 2 mRNA. In addition, lactacystin dysregulated mitogen-activated protein kinase phosphorylation in LPS-stimulated macrophages, but failed to inhibit IL-1 receptor-associated kinase-1 activity. Importantly, lactacystin also prevented LPS-induced shock in mice. These data strongly suggest that the proteasome complex regulates the LPS-induced signal transduction and that it may be an important therapeutic target in Gram-negative sepsis.

That TLRs share a common MyD88-dependent signaling pathway which results in the generation of nuclear DNA-binding proteins, such as NF-kappaB, is a well-accepted paradigm. However, studies from our laboratories and others suggested that TLR4 agonists elicit a more diverse pattern of gene expression in murine macrophages than TLR2 agonists. The data presented show that activation of TLR4 by Escherichia coli LPS results in an MyD88-independent, TIRAP/Mal-dependent signaling pathway that, in turn, leads to early induction of interferon-beta (IFN-beta). IFN-beta, in turn, acts in an autocrine/paracrine fashion on the macrophage to activate STAT1-containing DNA binding complexes that participate in the induction of genes not expressed in response to natural or synthetic TLR2 agonists. These data support the hypothesis that the host response to microbes is controlled by TLRs at two levels: (i) the "sensing" of differences in microbial structures through the TLR extracellular domain; and (ii) signaling pathways that are initiated via interactions through unique intracytoplasmic regions of different TLRs with adaptor proteins.

Toll-like receptor 2 (TLR2) agonists induce a subset of TLR4-inducible proinflammatory genes, which suggests the use of differential signaling pathways. Murine macrophages stimulated with the TLR4 agonist Escherichia coli lipopolysaccharide (LPS), but not with TLR2 agonists, induced phosphorylation of signal transducer and activator of transcription 1alpha (STAT1alpha) and STAT1beta, which was blocked by antibodies to interferon beta (IFN-beta) but not IFN-alpha. All TLR2 agonists poorly induced IFN-beta, which is encoded by an immediate early LPS-inducible gene. Thus, the failure of TLR2 agonists to induce STAT1-dependent genes resulted, in part, from their inability to express IFN-beta. TLR4-induced IFN-beta mRNA was MyD88- and PKR (double-stranded RNA-dependent protein kinase)-independent, but TIRAP (Toll-interleukin 1 receptor domain-containing adapter protein)-dependent. Together, these findings provide the first mechanistic basis for differential patterns of gene expression activated by TLR4 and TLR2 agonists.

Qureshi, N.; Perera, P.-Y.; Shen, J.; Gao, J. J.; Dhar, A.; Vogel, S. N.; Morrison, D. C. Author Information

Using a panel of LPS-inducible genes, selected for the capacity of their products to contribute to endotoxicity, normal macrophages were compared to macrophages deficient in CD14, CD11b/CD18, or TLR4 to elicit gene expression in response to Escherichia coli LPS or the LPS mimetic, Taxol. All genes were TLR4-dependent. At low doses of LPS or Taxol, all genes were also CD14-dependent; however, IP-10 and ICSBP remained poorly inducible even at much higher concentrations. A distinct subset of genes (COX-2, IL-12 p40, and IL-12 p35) was CD11b/CD18-dependent. NF-κB translocation and MAPK phosphorylation were dysregulated in receptor-deficient macrophages. In contrast to E. coli LPS, a Porphyromonas gingivalis LPS preparation was found to be TLR2-, rather than TLR4-dependent, and resulted in differential expression of genes within the panel. These data suggest that: (i) TLR4 is necessary, but not sufficient, to induce the full repertoire of genes examined; (ii) CD14 and CD11b/CD18 facilitate signaling for induction of select subsets of genes that are also TLR4-dependent; and (iii) signaling through TLR2 versus TLR4 differs quantitatively/qualitatively. These data support an LPS signaling complex on murine macrophages that minimally includes CD14, CD11b/CD18, and TLR4 to respond to E. coli LPS to elicit the full spectrum of gene expression.

Overproduction of inflammatory mediators by macrophages in response to Gram-negative LPS has been implicated in septic shock. Recent reports indicate that three membrane-associated proteins, CD14, CD11b/CD18, and Toll-like receptor (TLR) 4, may serve as LPS recognition and/or signaling receptors in murine macrophages, Therefore, the relative contribution of these proteins in the induction of cyclooxygenase 2 (COX-2), IL-12 p35, IL-12 p40, TNF-alpha, IFN-inducible protein (IP)-10, and IFN consensus sequence binding protein (ICSBP) genes in response to LPS or the LPS-mimetic, Taxol, was examined using macrophages derived from mice deficient for these membrane-associated proteins. The panel of genes selected reflects diverse macrophage effector functions that contribute to the pathogenesis of septic shock. Induction of the entire panel of genes in response to low concentrations of LPS or Taxol requires the participation of both CD14 and TLR4, whereas high concentrations of LPS or Taxol elicit the expression of a subset of LPS-inducible genes in the absence of CD14, In contrast, for optimal induction of COX-2, IL-12 p35, and IL-12 p40 genes by low concentrations of LPS or by all concentrations of Taxol, CD11b/CD18 was also required. Mitigated induction of COX-2, IL-12 p35, and IL-12 p40 gene expression by CD11b/CD11-deficient macrophages correlated with a marked inhibition of NF-kappaB nuclear translocation and mitogen-activated protein kinase (MAPK) activation in response to Taxol and of NF-kappaB nuclear translocation in response to LPS, These findings suggest that for expression of a full repertoire of LPS-/Taxol-inducible genes, CD14, TLR4, and CD11b/CD18 must be coordinately engaged to deliver optimal signaling to the macrophage.

A point mutation in the Tlr4 gene, which encodes Toll-like receptor 4, has recently been proposed to underlie LPS hyporesponsiveness in C3H/HeJ mice (Lpsd). The data presented herein demonstrate that F1 progeny from crosses between mice that carry a approximately 9-cM deletion of chromosome 4 (including deletion of LpsTlr4) and C3H/HeJ mice (i.e., Lps0 x Lpsd F1 mice) exhibit a pattern of LPS sensitivity, measured by TNF activity, that is indistinguishable from that exhibited by Lpsn x Lpsd F1 progeny and whose average response is "intermediate" to parental responses. Thus, these data provide clear functional support for the hypothesis that the C3H/HeJ defect exerts a dominant negative effect on LPS sensitivity; however, expression of a normal Toll-like receptor 4 molecule is apparently not required.

The anti-tumor agent, paclitaxel (active ingredient of Taxol®), is best recognized for its ability to bind to microtubules and to block cell division. However, it has more recently been demonstrated to mimic the varied effects of bacterial lipopolysaccharide (LPS) in murine macrophages, actions that appear to be dissociable from its well-characterized β-tubulin binding capacity. Secretion of tumor necrosis factor alpha (TNFα) and induction of TNFα gene expression were assessed in macrophages treated with paclitaxel analogs. Two structural modifications resulted in elevated TNFα mRNA and protein secretion: (i) the presence of a cyclopropane carboxylate ester at C-4 rather than an acetate; or (ii) deoxygenation of the C-7 position. Certain modifications essentially eliminated activity: derivatization of the side chain 2′ hydroxy group to form an ethyl carbonate, the presence of a benzoate at C-4 rather than an acetate, or de-acetylization to leave a free hydroxy group at the C-10 position. Substitution of the phenyl group at the 3′ carbon position of the side chain with a 2-furyl group or the presence of a 2- or 3-pyridine carboxylate moiety at C-2 rather than a benzoate also resulted in a significant reduction in TNFα. These structure—activity relationships can be distinguished from those that affect the cytotoxic effects of paclitaxel which are attributable to its microtubule binding activity.

The anti-tumor agent, Taxol, is a potent LPS mimetic in murine macrophages, an activity that is dissociable from its well-characterized anti-mitotic activity which is mediated by microtubule hyperstabilization. A photoactivatable Taxol analog was used to identify components of a putative, shared LPS signaling apparatus in murine macrophage membranes. We report here that CD18, the β chain of the β2-integrin, Mac-1, represents a major Taxol binding protein in murine macrophages.

The antitumor agent, Taxol, shares with bacterial LPS the ability to activate murine macrophages, and its LPS-mimetic effects are blocked by LPS analogue antagonists. Since CD14 is central to the recognition of LPS by macrophages, we sought to examine a role for CD14 in the response to Taxol vs LPS. A comparison of responses of macrophages from wild-type mice with those from mice lacking CD14 due to a targeted disruption of the CD14 gene (CD14-deficient knockout (CD14KO)) revealed that like LPS, Taxol induces both CD14-dependent and -independent pathways of gene activation, although the CD14 dependency of Taxol stimulation is much less striking than that observed with LPS. The macrophage interaction with low concentrations of LPS (< or = 10 ng/ml) is largely CD14 dependent, as evidenced by the lack of induction of TNF-alpha, IL-1beta, and interferon-inducible protein-10 (IP-10) genes by CD14KO macrophages cultured in the absence of soluble CD14 (i.e., in autologous CD14KO -/- mouse serum). However, at high concentrations of LPS or Taxol, a CD14-independent pathway of activation is observed: this pathway leads to minimal IP-10 gene induction, even though induction of TNF-alpha and IL-1beta occurs. Measurements of TNF secretion followed a similar pattern to that observed at the level of steady state mRNA. These data suggest the existence of two pathways of activation by both LPS and Taxol: one that is CD14 dependent and leads to induction of TNF-alpha, IL-1beta, and IP-10 gene induction, and a CD14-independent pathway that results in the induction of TNF-alpha and IL-1beta, with minimal induction of IP-10.

ABSTRACTMonocytes/macrophages play a central role in mediating the effects of lipopolysaccharide (LPS) derived from gram-negative bacteria by the production of proinflammatory mediators. Recently, it was shown that the expression of cytokine genes for tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), and interferon-inducible protein-10 (IP-10) by murine macrophages in response to low concentrations of LPS is entirely CD14 dependent. In this report, we show that murine macrophages respond to low concentrations of LPS (≤2 ng/ml) in the complete absence of serum, leading to the induction of TNF-α and IL-1β genes. In contrast to the TNF-α and IL-1β genes, the IP-10 gene is poorly induced in the absence of serum. The addition of recombinant human soluble CD14 (rsCD14) had very little effect on the levels of serum-free, LPS-induced TNF-α, IL-1β, and IP-10 genes. In contrast, the addition of recombinant human LPS-binding protein (rLBP) had opposing effects on the LPS-induced TNF-α or IL-1β and IP-10 genes. rLBP inhibited LPS-induced TNF-α and IL-1β genes, while it reconstituted IP-10 gene expression to levels induced in the presence of serum. These results provide further evidence that the induction of TNF-α or IL-1β genes occurs via a pathway that is distinct from one that leads to the induction of the IP-10 gene and that the pathways diverge at the level of the initial interaction between LPS and cellular CD14. Additionally, the results presented here indicate that LPS structural analog antagonistsRhodobacter sphaeroidesdiphosphoryl lipid A and SDZ 880.431 are able to inhibit LPS-induced TNF-α and IL-1β in the absence of serum, while a synthetic analog ofRhodobacter capsulatuslipid A (B 975) requires both rsCD14 and rLBP to function as an inhibitor.

Interaction of bacterial lipopolysaccharide (LPS) with macrophages results in the induction of a cascade of cytokines that mediate the varied effects of LPS. An early intracellular signaling event that follows receptor engagement is the activation of transcription factor NF-kappaB. Nf-kappaB has been shown to be important for the induction of many LPS-inducible cytokine genes, including tumor necrosis factor alpha, interleukin-1beta, and interleukin-6. Previously, we and others have shown that the antitumor agent paclitaxel (Taxol) is able to mimic bacterial LPS in its ability to activate murine macrophages. In this report, we have extended these findings by demonstrating that paclitaxel, like LPS, is able to stimulate the translocation of primarily p50-p65 heterodimers of NF-kappaB to the nucleus. This activation is dose dependent and requires a concentration of > or =5 microM paclitaxel. The kinetics of NF-kappaB activation by paclitaxel are slower than those of LPS: by 15 min poststimulation, LPS-induced NF-kappaB activation was readily detected, whereas the paclitaxel-induced NF-kappaB activation was minimal. Moreover, paclitaxel- and protein-free LPS-induced translocation of NF-kappaB was seen only in macrophages derived from LPS-responsive C3H/OuJ mice and not from the LPS-hyporesponsive C3H/HeJ mice, a finding that is consistent with those of previous genetic studies linking paclitaxel responsiveness to the Lps gene. Finally, the LPS structural antagonist Rhodobacter sphaeroides diphosphoryl lipid A inhibited both LPS-and paclitaxel-induced NF-kappaB activation, suggesting a common receptor component in this activation.

LPS-stimulated macrophages produce cytokines which, at appropriate levels, direct successful immune responses against harmful pathogens. However, excessive cytokine production, as seen in endotoxemia, results in pathophysiological damage to the host. Therefore, understanding mechanisms of cytokine regulation may aid the development of strategies designed to control cytokine production during an ongoing immune response. We have examined the role of okadaic acid-sensitive phosphatases in the production of cytokines and nitric oxide by macrophages. Okadaic acid induces TNFα, IL-1β, IL-6, IFN-β, and IP-10, but not IL-10 or IL-12 (p40) mRNA. Okadaic acid differentially regulates the expression of LPS-inducible IL-10 and IL-12 (p40) mRNA. These findings suggest that okadaic acid-sensitive phosphatases are key regulators of cytokine production in unstimulated and immune-activated macrophages. Finally, okadaic acid inhibits iNOS mRNA and nitric oxide production by macrophages activated by LPS and IFN-γ.

Lipopolysaccharide (LPS) is a potent bacterial product that has been shown to act on many different cell types both in vivo and in vitro, Injection of immunologically competent mice with LPS results in increased serum cytokine levels, followed by an array of pathophysiologic alterations that can ultimately lead to death, In this study, we examined the response of severe combined immunodeficient (SCID) mice to LPS, These mice lack mature T and B cells and have been shown to be an important model for analyzing the contribution of innate immune responses to infectious agents, Injection of SCID mice with LPS resulted in increases in CSF, TNF, and IFN levels in serum that were similar to the responses of immunocompetent controls, In response to LPS, both SCID and control mice exhibited similar levels of hypoglycemia, LPS-induced toxicity was assessed in D(+)-galactosamine-sensitized animals, SCID mice were comparably sensitive to the lethal effects of LPS as control BALB/c mice. To assess the role of natural killer (NK) cells in LPS-induced cytokine responses, BALB/c and SCID mice were injected with anti-asialo-GM1 antibody prior to injection of LPS, No significant effect on LPS-induced CSF or blood glucose levels were seen, although NK-depleted SCID mice produced somewhat more IFN in response to LPS than normal mice. Thus, NK cells are not a major source of these early LPS-induced cytokines, These data suggest that mature T and B cells and NK cells do not contribute to the initial wave of cytokines produced in response to LPS, but may contribute as secondary producers of cytokines involved in the cytokine cascade elicited by LPS injection.

LPS-stimulated macrophages (M phi) produce inflammatory mediators that are largely responsible for the pathophysiology associated with septic shock. M phi respond to LPS with rapid protein phosphorylation and dephosphorylation on serine, threonine, and tyrosine residues. If these events are critical for the cellular response to LPS, the kinases and/or phosphatases involved may be vulnerable targets for pharmacologic intervention. Recent studies demonstrated that tyrosine kinase inhibitors block LPS-induced tyrosine phosphorylation of MAP kinases as well as TNF-alpha and IL-1 beta production. To investigate a role for serine/threonine phosphatases, we evaluated the effect of calyculin A, a potent serine/threonine phosphatase inhibitor, on LPS stimulation of murine M phi. Pretreatment of M phi with calyculin A inhibited LPS-induced expression of six immediate-early genes: TNF-alpha, IL-1 beta, IFN-beta, IP-10, IRF-1, and TNFR-2. Calyculin A added 1.5 h after LPS treatment greatly reduced accumulation of IP-10, IRF-1, and TNFR-2 mRNA, but not TNF-alpha, IL-1 beta, and IFN-beta mRNA. Calyculin A, in the absence or presence of LPS, resulted in sustained tyrosine phosphorylation of the MAP kinases. These findings suggest that an "early" serine/threonine phosphatase activity is essential for LPS stimulation of M phi and that the activation of MAP kinases is not sufficient for the induction of these immediate-early genes. The requirement for a "late" phosphatase activity for expression of a subset of LPS-inducible genes dissociates at least two regulatory pathways in LPS signal transduction.

The model in Figure 3 summarizes the data presented above. Using the induction of the select panel of LPS-inducible genes and the phosphorylation on tyrosine of specific MAP kinases, we have been able to dissociate three signaling pathways shared by LPS and its analogs and mimetics: a pathway that leads to tyrosine phosphorylation, one that leads to the induction of a gene subset including TNF alpha, TNFR-2, and IL-1 beta, and a pathway that results in induction of IP-10, D3, and D8 gene expression. It is still unclear if macrophage activation by non-LPS products occurs entirely through distinct yet redundant pathways or if other signaling receptors ultimately tie into the same intermediate pathways. This approach may identify particular stimuli as tools to induce specific pathways leading to select gene subsets and/or tyrosine kinase activation and, perhaps, identify a pathway deficient in C3H/HeJ macrophages.