Ligation of the α2-macroglobulin signaling receptor on macrophages induces synthesis of platelet activating factor

The binding of receptor-recognized forms of α₂-macroglobulin (α₂M) to macrophage α₂M signaling receptors increases inositol-1,4,5-triphosphate synthesis and induces Ca²⁺ mobilization. In this report, we demonstrate that ligation of the macrophage α₂M signaling receptor is also associated with synthesis of platelet activating factor (PAF) by both the de novo and remodeling pathways. Both α₂M-methylamine and a cloned and expressed 20-kDa receptor binding fragment (RBF) from rat α₂M+, stimulated macrophage synthesis of PAF from [³H]acetate, [³H]methylcholine, and 1-O-[³H]alkyl lyso-PAF by two- to threefold. PAF levels reached a peak in 20 min after the cells were exposed to α₂M-methylamine or RBF; they remained elevated for about 1 h after ligand addition to the cells. When [³H]methylcholine was the substrate, pertussis toxin did not block PAF synthesis, but the protein kinase C inhibitor staurosporin reduced synthesis by 65–70%. Cycloheximide completely abolished the increase in synthesis of PAF by macrophages exposed to α₂M-methylamine. By contrast, when [³H]acetate was employed as a precursor, staurosporin or cycloheximide did not abolish the increase in PAF synthesis. These studies suggest that protein kinase C is necessary for the induction of the de novo pathway by α₂M-methylamine. Both α₂M-methylamine and RBF stimulated the activity of lyso-PAF acetyltransferase by about fourfold. Both ligands also stimulated the activity of PAF acetylhydrolase by about six- to sevenfold, indicating that ligation of the α₂M signaling receptor also regulates the degradation of PAF. The ability of receptor-recognized forms of α₂M to regulate levels of PAF suggests that α₂M-proteinase complexes not only regulate macrophage function by activating intracellular signaling but also may indirectly regulate the function of other cells that cannot bind α₂M-proteinase complexes. © 1996 Wiley-Liss, Inc.     

A short note on predicted motifs in the Thaparocleidus wallagonius genome

Thaparocleidus wallagonius is a monogenean parasite and a fish-borne pathogen with a worldwide distribution. The genome for Thaparocleidus wallagonius is known. Therefore, it is of interest to report the DNA motif analysis data in the 18S rDNA of Thaparocleidus wallagonius collected from the fish Wallago attu in India. This data forms a framework for an in-depth analysis of the parasite biology and development, immune evasion strategies, virulence and long-term survival within the definitive host.     

Early postnatal cardiac changes and premature death in transgenic mice overexpressing a mutant form of serum response factor

Serum response factor (SRF) is a key regulator of a number of extracellular signal-regulated genes important for cell growth and differentiation. A form of the SRF gene with a double mutation (dmSRF) was generated. This mutation reduced the binding activity of SRF protein to the serum response element and reduced the capability of SRF to activate the atrial natriuretic factor promoter that contains the serum response element. Cardiac-specific overexpression of dmSRF attenuated the total SRF binding activity and resulted in remarkable morphologic changes in the heart of the transgenic mice. These mice had dilated atrial and ventricular chambers, and their ventricular wall thicknesses were only 1/2 to 1/3 the thickness of that of nontransgenic mice. Also these mice had smaller cardiac myocytes and had less myofibrils in their myocytes relative to nontransgenic mice. Altered gene expression and slight interstitial fibrosis were observed in the myocardium of the transgenic mice. All the transgenic mice died within the first 12 days after birth, because of the early onset of severe, dilated cardiomyopathy. These results indicate that dmSRF overexpression in the heart apparently alters cardiac gene expression and blocks normal postnatal cardiac growth and development.     

Dynamin inhibits phosphatidylinositol 3-kinase in hematopoietic cells

Phosphatidylinositol 3-kinase (PI 3-kinase) plays a role in late stages of endocytosis as well as in cellular proliferation and transformation. The SH3 domain of its regulatory p85 subunit stimulates the GTPase activity of dynamin in vitro. Dynamin is a GTPase enzyme required for endocytosis of activated growth factor receptors. An interaction between these proteins has not been demonstrated in vivo. Here, we report that dynamin associates with PI 3-kinase in hematopoietic cells. We detected both p85 and PI 3-kinase activity in dynamin immune complexes from IL-3-dependent BaF3 cells. However, this association was significantly reduced in BaF3 cells transformed with the BCR/abl oncogene. After transformation only a 4-fold increase in PI 3-kinase activity was detected in dynamin immune complexes, whereas grb2 associated activity was elevated 20-fold. Furthermore, dynamin inhibited the activity of both purified recombinant and immunoprecipitated PI 3-kinase. In BaF3 cells expressing a temperature-sensitive mutant of BCR/abl, a significant decrease in p85 and dynamin association was observed 4 h after the induction of BCR/abl activity. In contrast, in IL-3-stimulated parental BaF3 cells, this association was increased. Our results demonstrate an in vivo association of PI 3-kinase with dynamin and this interaction regulates the activity of PI 3-kinase.     

The TolC protein of Escherichia coli serves as a cell-surface receptor for the newly characterized TLS bacteriophage

The TolC protein of Escherichia coli is implicated in a variety of diverse cellular functions, including antibiotic efflux and alpha-hemolysin secretion. An incidental role of TolC is to facilitate the entry of the bacteriophage TLS and colicin E1 into the bacterial cell. Despite the resolution of TolC's atomic structure, the roles of specific residues in its diverse functions are unknown. Here, we describe a genetic strategy for isolating missense tolC mutations that abolish the bacteriophage receptor activity of the TolC protein without influencing its role in antibiotic efflux. These spontaneous mutations affected two regions of the TolC protein and included base-pair substitutions, insertions, and deletions. Comparison of the TolC sequence with those of its homologues revealed two hypervariable stretches that were predicted to represent loops. Interestingly, all but one of the TolC alterations preventing phage binding were located in these two hypervariable regions, which are likely to be exposed on the cell surface. This was substantiated by the recently solved three-dimensional structure of TolC. Curiously, all the phage-resistant TolC mutants showed varying degrees of resistance to colicin E1, suggesting the involvement of overlapping regions of TolC in colicin E1 import and phage binding.The phage used in this study, TLS, was earlier reported as a strain of U3. However, we show here that, unlike the previously reported lipopolysaccharide-specific U3 phage, this phage displays a distinctly different host range and discrete morphological features and, in addition to utilizing TolC as receptor, it requires the inner core of a lipopolysaccharide.     

Liposomes of terbutaline sulphate: in vitro and in vivo studies

In vitro studies were conducted to understand the comparative drug diffusion pattern, across artificial membrane, of the drug and of the prepared liposomes of different liposomal membrane composition. In vivo studies were carried out to determine the extent and time-course of pulmonary tissue uptake of administered liposomes containing terbutaline sulphate(TER) on rat lungs. In vitro studies revealed that the drug released from the prepared liposomes obeys Higuchi's diffusion controlled model. Different loading doses and release patterns of drug from the liposomes can be obtained by altering the PC:CHOL ratio and incorporation of cholesterol was found to reduce permeability of the membrane. Similarly drug absorption in vivo in rat's lung following intratracheal instillation, prolonged over 12 hr by liposomal entrapment of TER. The findings of present investigation indicated that liposomally encapsulated TER can be used for pulmonary delivery for maximizing the therapeutic efficacy and reducing undesirable side effects.     

Upregulation of macrophage plasma membrane and nuclear phospholipase D activity on ligation of the alpha2-macroglobulin signaling receptor: involvement of heterotrimeric and monomeric G proteins

The effect of ligating the alpha2-macroglobulin signaling receptor (alpha2MSR) with receptor-recognized forms of alpha2M (alpha2M*) was studied with respect to phospholipase D (PLD) activity in murine macrophages, their plasma membranes, and nuclei. PLD activity in plasma membranes and nuclei increased linearly up to a ligand concentration of about 100 pM of either alpha2M* or a cloned and expressed receptor binding fragment (RBF). The RBF binding site mutant K1370A, which binds with high affinity to alpha2MSR, also increased nuclear PLD activity comparable to RBF and alpha2M*. Phorbol dibutyrate caused a two- to threefold stimulation of membrane and nuclear PLD activity, whereas PLD activity was nearly abolished by downregulation of protein kinase C; prior treatment with staurosporin, genestein, cyclosporin A, actinomycin D; or chelation of intracellular Ca2+. In permeabilized macrophages, isolated plasma membranes, and nuclei, GTP-gamma-S increased alpha2M*-stimulated PLD activity via a pertussis toxin-insensitive G protein and this effect was abolished on preincubation with GDP-beta-S. Incubation of plasma membranes with polyclonal antibody against sARFII, or the addition of cytosol which was immunoprecipitated with antibody against sARFII, greatly reduced alpha2M*-stimulated PLD activity in the presence of GTP-gamma-S. Preincubation of plasma membranes with GDP-beta-S prior to the addition of GTP-gamma-S and recombinant ARF1 significantly inhibited alpha2M*-stimulation of PLD activity. Nuclear PLD activity was maximally stimulated in the presence of both GTP-gamma-S and rARF1, whereas plasma membrane PLD activity was maximally stimulated in the presence of rARF1, GTP-gamma-S, RhoA, and ATP. In contrast, nuclear PLD activity was not affected by RhoA either alone or in combination with GTP-gamma-S or ATP.     

Chemical fingerprinting of Andrographis paniculata using HPLC, HPTLC and densitometry

An attempt has been made to develop a method by which to determine the chemical fingerprint of Andrographis paniculata (Acanthaceae). High-performance thin layer chromatography (HPTLC) was used to analyse hexane, chloroform, methanol and water extracts of leaves of A. paniculata. A computerised densitometer was applied to the two-dimensional spectrographic image analysis of the HPTLC plates. An HPLC equipped with a photodiode array detector was used for the analyses of these different extracts. The analyses showed that andrographolide and neoandrographolide are absent in the hexane extract but are present in greater amounts in the methanol extract as compared with the other extracts. These chromatograms may serve as a chemical fingerprint of the drug A. paniculata for quality control purposes and in the preparation of formulations based on the drug.