Physicochemical evidence that Treponema pallidum TroA is a zinc-containing metalloprotein that lacks porin-like structure.
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Although TroA (Tromp1) was initially reported to be a Treponema pallidum outer membrane protein with porin-like properties, subsequent studies have suggested that it actually is a periplasmic substrate-binding protein involved in the transport of metals across the treponemal cytoplasmic membrane. Here we conducted additional physicochemical studies to address the divergent viewpoints concerning this protein. Triton X-114 phase partitioning of recombinant TroA constructs with or without a signal sequence corroborated our prior contention that the native protein's amphiphilic behavior is due to its uncleaved leader peptide. Whereas typical porins are trimers with extensive beta-barrel structure, size exclusion chromatography and circular dichroism spectroscopy revealed that TroA was a monomer and predominantly alpha-helical. Neutron activation, atomic absorption spectroscopy, and anomalous X-ray scattering all demonstrated that TroA binds zinc in a 1:1 molar stoichiometric ratio. TroA does not appear to possess structural features consistent with those of bacterial porins. (+info)
Heat treatment of normal human sera reveals antibodies to bactericidal permeability-inducing protein (BPI).
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Heat treatment of normal sera to 56 degrees C for 30 min, a common procedure for the inactivation of viruses, e.g. HIV, reveals the presence of antibodies to neutrophil cytoplasm antigens (ANCA), as detected by indirect immunofluorescence on ethanol-fixed human neutrophils and by antigen-specific ELISA for BPI. Reactivity was not seen to the other common vasculitis-associated antigens proteinase 3 (PR3) or myeloperoxidase (MPO). The effect of temperature was maximal at 56 degrees C, with substantial antibody demonstrable after only 5 min at this temperature. In experiments using polyethylene glycol (PEG)6000 to remove immune complexes, the effect of heating could be abrogated by preincubation with 8% PEG, which suggested that these anti BPI antibodies might be complexed in sera. After passage of normal plasma over a protein G column, the acid-eluted fraction contained elevated levels of antibodies to BPI but not to other vasculitis-associated antigens such as PR3 or MPO, nor to glomerular basement membrane (GBM), the Goodpasture antigen which is recognized by the pathogenically important human antibodies shown to mediate nephritis in transfer experiments. Moreover the levels of anti-BPI in the IgG fraction could be augmented by preincubation with glycine pH 2.5 for 30 min. This anti-BPI activity could be inhibited by addition of the unbound material from the protein G column and this inhibitory material was not heat-labile at 56 degrees C. The molecular specificity of this autoreactivity was confirmed using recombinant BPI in coincubation experiments and the epitope localized to the C or N terminal moieties by the use of recombinant fusion proteins. (+info)
Localization of actin in Moloney murine leukemia virus by immunoelectron microscopy.
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Immunoelectron microscopy was used to detect actin in wild-type (wt) Moloney murine leukemia virus (MoMuLV) and in virus-like particles (VLP) produced by recombinant Semliki Forest virus expressing only the MoMuLV gag polyprotein. Gold immunolabeling revealed the presence of actin on the surface of delipidized VLP and delipidized wt virus particles. Statistical evaluation of the number of colloidal gold particles per VLP revealed a large range of values and a prevalence of VLP with small numbers of gold particles. Labeling for actin was lost after prolonged treatment of VLP with 1% Nonidet-P40, high-pH buffer, or gelsolin. Gold immunolabeling with antibodies to gag proteins p15 (MA) and p12 and p30 (CA) was abundant and was not affected by treatment of VLP or wt virus with 1% Nonidet or gelsolin. VLP treated with a mixture of detergent and aldehyde fixatives showed more uniform and consistent labeling for actin than without fixatives. Negative staining or heavy metal shadowing revealed a globular surface of delipidized VLP. Stereomicrographs of gold-immunolabeled VLP showed that p15gag and p12gag were associated with the globular projections. Delipidized VLP were also well labeled with antibody to p30gag, which indicated that the gag shell permitted access of antibodies to p30gag and was therefore not a closely packed structure. Labeling for actin-binding proteins moesin and ezrin was negative in both the wt virus and the VLP. The absence of Gaussian distribution of actin in the sample of VLP suggests that actin is not a structural protein and its presence in MuLV virus particles may be fortuitous. This, however, does not rule out any possible role of actin in transport, assembly, budding, or release of virus particles, events which take place in the cytoplasm or at the plasma membrane. The site of actin in VLP is discussed in relation to the present knowledge of the molecular organization of the MuLV gag shell. (+info)
Previous work has shown that plasmid DNA can be encapsulated in small 'stabilized plasmid-lipid particles' (SPLP) composed of 1, 2-dioleyl-3-phosphatidylethanolamine (DOPE), the cationic lipid N, N-dioleyl-N,N-dimethylammonium chloride (DODAC) and poly(ethylene glycol) (PEG) conjugated ceramides (PEG-Cer), employing a detergent dialysis procedure. These SPLP have potential as vectors for in vivo gene therapy. This study is aimed at characterizing the influence of the cationic lipid and PEG-Cer species on SPLP formation and in vitro transfection properties. It is shown that the transfection potency of SPLP is sensitive to the cationic lipid species employed, the size of the PEG polymer incorporated in the PEG-ceramide and the length of the acyl chain contained in the ceramide anchor. With regard to the influence of cationic lipid, the transfection levels achieved were highest for SPLP containing N-[2, 3-(dioleyloxy)propyl]-N,N-dimethyl-N-cyanomethylammonium chloride (DODMA-AN) and lowest for SPLP containing 3-beta-[N-(N', N'-dimethylaminoethyl)carbamoyl]-cholesterol (DC-CHOL), according to the series DODMA-AN>N-[2,3-(dioleyloxy)propyl]-N,N, N-trimethylammonium chloride (DOTMA)>DODAC>N,N-distearyl-N, N-dimethylammonium chloride (DSDAC)>DC-CHOL. Incorporation of short (PEG(750)) PEG polymers in the PEG-ceramide components resulted in modest improvements in transfection levels over PEG(2000) and PEG(5000) polymers, however variation of the length of the acyl chain contained in the hydrophobic ceramide anchor from octanoyl (PEG-CerC(8)) to myristoyl (PEG-CerC(14)) to arachidoyl (PEG-CerC(20)) had the most dramatic effects. Transfection levels achieved for SPLP containing PEG-CerC(8) were substantially larger than observed for SPLP containing PEG-CerC(14) or PEG-CerC(20), consistent with a requirement for the PEG-ceramide to dissociate from the SPLP surface for maximum transfection potency. It is also shown that the ability of SPLP to be accumulated into cells is a dominant factor influencing transfection potency, and that the transfection potency of SPLP that are accumulated is at least equivalent to that of cationic lipid-plasmid DNA complexes. (+info)
Intravenous administration of superoxide dismutase entrapped in long circulating liposomes. II. In vivo fate in a rat model of adjuvant arthritis.
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Rheumatoid arthritis (RA) is a prevalent and debilitating autoimmune disease that affects the joints. RA is characterized by an infiltration of the affected joint by blood-derived cells. In response to activation, these cells generate reactive oxygen species, resulting in an oxidative stress situation. One approach to counteract this oxidative stress situation is the use of antioxidants as therapeutic agents. The free radical scavenger enzyme superoxide dismutase (SOD) may be used as a therapeutic agent in rheumatoid arthritis, but its rapid elimination from the circulation is a major limitation. Targeted delivery of SOD may overcome this limitation. In this study, the utility of PEGylated liposomes (PEG-liposomes) for targeting SOD to arthritic sites was explored. The targeting of SOD to arthritic sites following intravenous administration of both PEG-liposomes and positively charged liposomes lacking PEG but containing stearylamine (SA-liposomes) in rats with adjuvant arthritis was studied. At 24 h post injection, the blood levels of long circulating liposomes with a mean size of 0.11 micrometer and 0.20 micrometer were 8- and 3-fold higher, respectively, as compared to the SA-liposomes. The majority of SOD administered in liposomal form remains within the liposomes when they circulate in the bloodstream. The highest target uptake was observed with PEG-liposomes with a mean size of 0.11 micrometer and the lowest uptake with the SA-liposomes. These results demonstrate that SOD can be targeted to inflamed sites most efficiently via small-sized PEG-liposomes. Small-sized PEG-coated liposomes are to be preferred if prolonged circulation and enhanced localization of SOD at arthritic sites are desired. (+info)
Water-soluble aluminium phthalocyanine-polymer conjugates for PDT: photodynamic activities and pharmacokinetics in tumour-bearing mice.
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The potential use of unsubstituted aluminium phthalocyanine (AlClPc) as a sensitizer for photodynamic therapy (PDT) of cancer has not been fully exploited in spite of its higher efficiency as compared to the sulphonated derivatives. This is largely due to the strong hydrophobic character of AlClPc which renders the material difficult to formulate for in vivo administration. We prepared two water-soluble derivatives of AlClPc by axial coordination of polyethyleneglycol (PEG, MW 2000) or polyvinylalcohol (PVA, MW 13,000-23,000) to the central aluminium ion. Their photodynamic activities were evaluated in vitro against the EMT-6 mouse mammary tumour cells and in vivo against the EMT-6 and the colon carcinoma Colo-26 tumours implanted intradermally in Balb/c mice. Pharmacokinetics were studied in the EMT-6 tumour-bearing mice. After 1 h incubation, the light dose required to kill 90% of cells (LD90) was at least three times less for AlClPc (Cremophor emulsion) as compared to AlPc-PEG and AlPc-PVA, while after 24 h incubation all three preparations were highly phototoxic. All three dye preparations induced complete EMT-6 tumour regression in 75-100% of animals at a low drug dose (0.25 micromol kg(-1)) following PDT (400 J cm(-2), 650-700 nm) at 24 h pi. Complete tumour regression in the Colo-26 tumour model was obtained in 30% of mice at a dose of 2 micromol kg(-1). In the non-cured animals, AlPc-PVA induced the most significant tumour growth delay. This dye showed a prolonged plasma half-life (6.8 h) as compared to AlClPc (2.6 h) and AlPc-PEG (23 min), lower retention by liver and spleen and higher tumour-to-skin and tumour-to-muscle ratios. Our data demonstrate that addition of hydrophilic axial ligands to AlPc, while modifying in vitro and in vivo kinetics, does not reduce the PDT efficiency of the parent molecule. Moreover, in the case of the polyvinylalcohol derivative, axial coordination confers advantageous pharmacokinetics to AlPc, which makes this photosensitizer a valuable, water soluble candidate drug for clinical PDT of cancer. (+info)
Structural equivalents of latency for lysosome hydrolases.
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1. Structure-linked latency, a trait for most lysosome hydrolase activities, is customarily ascribed to the permeability-barrier function performed by the particle-limiting membrane, which shields enzyme sites from externally added substrates. 2. The influence of various substrate concentrations on the reaction rate has been measured for both free (non-latent) and total (completely unmasked by Triton X-100) hydrolase activities in rat liver cell-free preparations. The substrates were: beta-glycerophosphate, phenolphthalein mono-beta-glucuronide. p-nitrophenyl N-acetyl-beta-D-glucosaminide and p-nitrophenyl beta-D-galactopyranoside. The ratio (free activity/total activity) X 100 is called fractional free activity at any given substrate concentration. 3. The fractional free activity of beta-glucuronidase and beta-N-acetylglucosaminidase were clearly independent of substrate concentration, over the range examined, in both homogenates and lysosome-rich fractions. The fractional free activity of acid phosphatase appeared to be either unaffected (homogenate) or even depressed (lysosome-rich fraction) by increasing the beta-glycerophosphate concentration. The fractional free activity of beta-galactosidase consistently showed a non-linear increase with increasing substrate concentration in both homogenates and lysosome-rich fractions. 4. Procedures such as treatment with digitonin, hypo-osmotic shock and acid autolysis, although effective in causing varying degrees of resolution of the latency of lysosome hydrolase activities, were unable to modify appreciably the pattern of dependence or independence of their fractional free activities on substrate concentration, as compared with that exhibited by control preparations. Ouabain did not affect the free beta-N-acetylglucosaminidase activity of liver homogenates at all. 5. Preincubation of control preparations with beta-glycerophosphate or p-nitrophenyl beta-galactoside did not result in any significant stimulation of the free hydrolytic activity toward these substrates. 6. The results consistently support the view that the membrane of "intact" lysosomes is virtually impermeable to all the substrates tested, except for p-nitrophenyl beta-galactoside, for which the evidence is contradictory. Moreover the progressive unmasking of the hydrolase activities produced by these procedures in vitro reflects the increasing proportion of enzyme sites that are fully accessible to their substrates rather than a graded increase in the permeability of the lysosomal membrane. (+info)
Suppression of induction of experimental immune mediated blepharoconjunctivitis by tolerogenic conjugates of the antigen and monomethoxypolyethylene glycol.
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AIM: Covalent conjugates consisting of diverse antigens coupled to optimal numbers of monomethoxypolyethylene glycol (mPEG) molecules have been shown to suppress antigen specific antibody formation. In this study, the possibility was examined that the same conjugates might prevent experimental immune mediated blepharoconjunctivitis (EC, formerly EAC) which had been shown to be caused by CD4(+) T cells-that is, to cell mediated immunity. METHODS: 6-8 week old male Lewis rats were used. The test groups of rats received two intravenous injections, each of 300 microg, of a conjugate of ovalbumin mPEG (OVA(mPEG)(11)) in phosphate buffered saline (PBS), 14 and 28 days before the single immunisation with OVA in complete Freund's adjuvant. The rats were challenged 3 weeks later by eye drops containing OVA; 24 hours later they were sacrificed, and their eyes, blood, and lymph nodes were harvested for histological examination and determination of anti-OVA antibody titres and levels of cellular immunity. Two control groups received PBS or OVA in PBS before immunisation. Furthermore, the possibility that OVA(mPEG)(11) may have induced OVA specific suppressor cells was tested by establishing the effects of the co-transfer of splenocytes from OVA(mPEG)(11) treated rats with OVA primed lymph node cells on the manifestations of EC. RESULTS: Either PBS or OVA pretreated rats, which had not received OVA(mPEG)(11), developed high levels of antibodies and cell mediated immune responses to OVA, and application of eye drops led to blepharoconjunctivitis with massive cellular infiltration. In contrast, pretreatment with OVA(mPEG)(11) prevented cellular infiltration into the lids and conjunctivas, as well as the formation of detectable humoral and cellular immunity against OVA. Co-transfer of splenocytes from OVA(mPEG)(11) treated rats with OVA primed lymph node cells suppressed the cellular infiltration on application of OVA on the conjunctiva. CONCLUSIONS: These data indicate that intravenous injection of OVA(mPEG)(11) conjugates suppressed both humoral and cellular immunity by the effects of antigen specific suppressor cells, thus leading to the inhibition of development of EC. (+info)