Fast Protein Liquid Chromatography Fractions Research Articles

Large-scale identification of calcium oxalate stone inhibitory proteins in normal human urine

Recent evidence has shown that proteins in normal human urine can inhibit calcium oxalate (CaOx) kidney stone formation. Herein, we performed fast protein liquid chromatography (FPLC) to fractionate normal human urinary proteins using anion-exchange (DEAE) and size-exclusion (Superdex 200) materials. FPLC fractions (F1-F15) were examined by CaOx crystallization, growth, aggregation and crystal-cell adhesion assays. The fractions with potent inhibitory activities against CaOx crystals were then subjected to mass spectrometric protein identification. The data revealed that 13 of 15 fractions showed inhibitory activities in at least one crystal assay. Integrating CaOx inhibitory scores demonstrated that F6, F7 and F8 had the most potent inhibitory activities. NanoLC-ESI-Qq-TOF MS/MS identified 105, 93 and 53 proteins in F6, F7 and F8, respectively. Among them, 60 were found in at least two fractions and/or listed among known inhibitors with solid experimental evidence in the StoneMod database (https://www.stonemod.org). Interestingly, 10 of these 60 potential inhibitors have been reported with lower urinary levels in CaOx stone formers compared with healthy (non-stone) individuals, strengthening their roles as potent CaOx stone inhibitors. Our study provides the largest dataset of potential CaOx stone inhibitory proteins that will be useful for further elucidations of stone-forming mechanisms and ultimately for therapeutic/preventive applications.

Protein fractionation and shotgun proteomics analysis of enriched bacterial cultures shed new light on the enzymatically catalyzed degradation of acesulfame

The removal of organic micropollutants in municipal wastewater treatment is an extensively studied field of research, but the underlying enzymatic processes have only been elucidated to a small extent so far. In order to shed more light on the enzymatic degradation of the artificial sweetener acesulfame (ACE) in this context, we enriched two bacterial taxa which were not yet described to be involved in the degradation of ACE, an unknown Chelatococcus species and Ensifer adhaerens, by incubating activated sludge in chemically defined media containing ACE as sole carbon source. Cell-free lysates were extracted, spiked with ACE and analyzed via target LC-MS/MS, demonstrating for the first time enzymatically catalyzed ACE degradation outside of living cells. Fractionation of the lysate via two-dimensional fast protein liquid chromatography (FPLC) succeeded in a partial separation of the enzymes catalyzing the initial transformation reaction of ACE from those catalyzing the further transformation pathway. Thereby, an accumulation of the intermediate transformation product acetoacetamide-n-sulfonic acid (ANSA) in the ACE-degrading fractions was achieved, providing first quantitative evidence that the cleavage of the sulfuric ester moiety of ACE is the initial transformation step. The metaproteome of the enrichments was analyzed in the FPLC fractions and in the unfractionated lysate, using shotgun proteomics via UHPLC-HRMS/MS and label-free quantification. The comparison of protein abundances in the FPLC fractions to the corresponding ACE degradation rates revealed a metallo-β-lactamase fold metallo-hydrolase as most probable candidate for the enzyme catalyzing the initial transformation from ACE to ANSA. This enzyme was by far the most abundant of all detected proteins and amounted to a relative protein abundance of 91% in the most active fraction after the second fractionation step. Moreover, the analysis of the unfractionated lysate resulted in a list of further proteins possibly involved in the transformation of ACE, most striking a highly abundant amidase likely catalyzing the further transformation of ANSA, and an ABC transporter substrate-binding protein that may be involved in the uptake of ACE into the cell.

Characterization and FPLC Analysis of Garbage Enzyme: Biocatalytic and Antimicrobial Activity

Fermentation of fruit and vegetable peels produces garbage enzyme (GE) which was used to obtain industrially beneficial products. This study was conducted for the characterization of GE and evaluation of its biocatalytic and antimicrobial activity. The maximum activity of amylase, protease and lipase were 1.782 units/mg, 0.050 units/mg and 0.355 units/mg respectively were found in optimum conditions which also showed antioxidant properties. The enzyme solution was separated by Fast Protein Liquid Chromatography (FPLC) and experiments were conducted for antimicrobial activity with the FPLC generated fractions of GE on both gram positive and gram negative bacteria. Inhibition zones were measured from concentration dependent bioassay analysis of lyophilized FPLC fractions. FPLC fraction II offered best zone of inhibition in Escherichia coli, Staphylococcus aureus and in Rhizobium leguminosarum. Molecular weight of above fraction was determined by SDS-PAGE technique. Significant research findings of this research work will help in developing newer potential commercializable antimicrobial agents from GE generated from fruit and vegetable waste.

Altered High Density Lipoprotein Composition in Behavioral Variant Frontotemporal Dementia.

Frontotemporal dementia (FTD) is a common cause of early onset dementia with behavioral variant FTD (bvFTD) being the most common form. bvFTD is characterized clinically by behavioral and personality changes, eating abnormalities, and pathologically, by systemic lipid dysregulation that impacts on survival. As lipoprotein metabolism is at the core of lipid dysregulation, here, we analyzed the composition, both proteins and lipids, of the two major lipoprotein classes in blood – high density lipoproteins (HDLs) and low density lipoproteins (LDLs). Fasted plasmas from bvFTD and Alzheimer’s disease (AD) patients and controls were fractionated using fast protein liquid chromatography (FPLC) and samples analyzed by lipid assays, ELISA and western blotting. We found that apolipoprotein A-I (apoA-I) and apolipoprotein A-II (apoA-II) levels in HDLs were decreased in bvFTD compared to controls, whereas apolipoprotein B (apoB) levels in LDLs were unaltered. We also found that cholesterol and triglyceride levels in FPLC fractions were altered in bvFTD compared to controls. The apoB:apoA-I ratio and the standard lipid ratios were significantly increased in bvFTD compared to AD and controls. Furthermore, we found that plasma apolipoprotein C-I and paraoxonase 1 levels were significantly altered in bvFTD and AD, respectively, compared controls. This study represents the first apolipoprotein analysis of bvFTD, and our results suggest altered HDL function and elevated cardiovascular disease risk in bvFTD.

Antimicrobial activity in the cuticle of the American lobster, Homarus americanus

American lobster, Homarus americanus, continues to be an ecologically and socioeconomically important species despite a severe decline in catches from Southern New England and Long Island Sound (USA) and a high prevalence of epizootic shell disease in these populations. A better understanding of lobster immune defenses remains necessary. Cuticle material collected from Long Island Sound lobsters was found to be active against a broad spectrum of bacteria, including Gram-negative and -positive species. The antimicrobial activity was characterized by boiling, muffling, and size fractioning. Boiling did not significantly reduce activity, while muffling did have a significant effect, suggesting that the active component is organic and heat stable. Size fractioning with 3 and 10 kDa filters did not significantly affect activity. Fast protein liquid chromatography fractions were also tested for antimicrobial activity, and fractions exhibiting protein peaks remained active. MALDI mass spectrometry revealed peptide peaks at 1.6, 2.8, 4.6, and 5.6 kDa. The data presented suggest that one or several antimicrobial peptides contribute to antimicrobial activity present in the American lobster cuticle.

Modulation of lipid metabolism with the overexpression of NPC1L1 in mouse liver

Niemann-Pick C1-like 1 protein (NPC1L1), a transporter crucial in intestinal cholesterol absorption, is expressed in human liver but not in murine liver. To elucidate the role of hepatic NPC1L1 on lipid metabolism, we overexpressed NPC1L1 in murine liver utilizing adenovirus-mediated gene transfer. C57BL/6 mice, fed on normal chow with or without ezetimibe, were injected with NPC1L1 adenovirus (L1-mice) or control virus (Null-mice), and lipid analyses were performed five days after the injection. The plasma cholesterol levels increased in L1-mice, and FPLC analyses revealed increased cholesterol contents in large HDL lipoprotein fractions. These fractions, which showed α-mobility on agarose electrophoresis, were rich in apoE and free cholesterol. These lipoprotein changes were partially inhibited by ezetimibe treatment and were not observed in apoE-deficient mice. In addition, plasma and VLDL triglyceride (TG) levels decreased in L1-mice. The expression of microsomal triglyceride transfer protein (MTP) was markedly decreased in L1-mice, accompanied by the reduced protein levels of forkhead box protein O1 (FoxO1). These changes were not observed in mice with increased hepatic de novo cholesterol synthesis. These data demonstrate that cholesterol absorbed through NPC1L1 plays a distinct role in cellular and plasma lipid metabolism, such as the appearance of apoE-rich lipoproteins and the diminished VLDL-TG secretion.

Establishment of a Transgenic Mouse Model Specifically Expressing Human Serum Amyloid A in Adipose Tissue

Obesity and obesity co-morbidities are associated with a low grade inflammation and elevated serum levels of acute phase proteins, including serum amyloid A (SAA). In the non-acute phase in humans, adipocytes are major producers of SAA but the function of adipocyte-derived SAA is unknown. To clarify the role of adipocyte-derived SAA, a transgenic mouse model expressing human SAA1 (hSAA) in adipocytes was established. hSAA expression was analysed using real-time PCR analysis. Male animals were challenged with a high fat (HF) diet. Plasma samples were subjected to fast protein liquid chromatography (FPLC) separation. hSAA, cholesterol and triglyceride content were measured in plasma and in FPLC fractions. Real-time PCR analysis confirmed an adipose tissue-specific hSAA gene expression. Moreover, the hSAA gene expression was not influenced by HF diet. However, hSAA plasma levels in HF fed animals (37.7±4.0 µg/mL, n = 7) were increased compared to those in normal chow fed animals (4.8±0.5 µg/mL, n = 10; p<0.001), and plasma levels in the two groups were in the same ranges as in obese and lean human subjects, respectively. In FPLC separated plasma samples, the concentration of hSAA peaked in high-density lipoprotein (HDL) containing fractions. In addition, cholesterol distribution over the different lipoprotein subfractions as assessed by FPLC analysis was similar within the two experimental groups. The established transgenic mouse model demonstrates that adipose tissue produced hSAA enters the circulation, resulting in elevated plasma levels of hSAA. This new model will enable further studies of metabolic effects of adipose tissue-derived SAA.

Amyloid-β42 Interacts Mainly with Insoluble Prion Protein in the Alzheimer Brain

The prion protein (PrP) is best known for its association with prion diseases. However, a controversial new role for PrP in Alzheimer disease (AD) has recently emerged. In vitro studies and mouse models of AD suggest that PrP may be involved in AD pathogenesis through a highly specific interaction with amyloid-β (Aβ42) oligomers. Immobilized recombinant human PrP (huPrP) also exhibited high affinity and specificity for Aβ42 oligomers. Here we report the novel finding that aggregated forms of huPrP and Aβ42 are co-purified from AD brain extracts. Moreover, an anti-PrP antibody and an agent that specifically binds to insoluble PrP (iPrP) co-precipitate insoluble Aβ from human AD brain. Finally, using peptide membrane arrays of 99 13-mer peptides that span the entire sequence of mature huPrP, two distinct types of Aβ binding sites on huPrP are identified in vitro. One specifically binds to Aβ42 and the other binds to both Aβ42 and Aβ40. Notably, Aβ42-specific binding sites are localized predominantly in the octapeptide repeat region, whereas sites that bind both Aβ40 and Aβ42 are mainly in the extreme N-terminal or C-terminal domains of PrP. Our study suggests that iPrP is the major PrP species that interacts with insoluble Aβ42 in vivo. Although this work indicated the interaction of Aβ42 with huPrP in the AD brain, the pathophysiological relevance of the iPrP/Aβ42 interaction remains to be established.

Oxidative Stress and Compartment of Gene Expression Determine Proatherosclerotic Effects of Inducible Nitric Oxide Synthase

Genetic and pharmacological inhibition of inducible nitric oxide synthase (iNOS) decreases atherosclerosis development. Potential proatherogenic effects of iNOS include iNOS mediated oxidative stress and iNOS expression in different cellular compartments. Lesional iNOS can potentially produce nitric oxide radicals (NO), superoxide radicals (O2(-)), or both; these radicals may then react to form peroxynitrite. Alternatively, O2(-) radicals from oxidases co-expressed with iNOS could react with NO to produce peroxynitrite. Therefore, the expression profiles of the genes that modulate the redox system in different iNOS-expressing cell compartments may determine the role of iNOS in atherosclerosis. We used apoE (apoE(-/-)) and apoE/iNOS double knockout (apoE(-/-)/ iNOS(-/-)) mice to assess vascular NO, O2(-), and peroxynitrite formation by electron spin resonance spectroscopy, high performance liquid chromatography, and 3-nitrotyrosine staining. The relevance of the iNOS expressing cell compartment was tested by bone marrow transplantation. We show that iNOS significantly contributes to vascular NO production and itself produces O2(-), leading to peroxynitrite formation in atherosclerotic lesions. Our bone marrow transplantation experiments show that bone marrow derived cells exclusively mediate the proatherosclerotic effects of iNOS in males, while both parenchymal and bone marrow derived iNOS equally contribute to atherosclerosis in females. Moreover, iNOS expression affects vascular remodeling. These findings establish for the first time that the proatherosclerotic effects of iNOS vary with sex in addition to the compartment of its expression.

Phospholipid transfer protein deficiency ameliorates diet-induced hypercholesterolemia and inflammation in mice

Phospholipid transfer protein (PLTP) facilitates the transfer of phospholipids from triglyceride-rich lipoproteins into HDL. PLTP has been shown to be an important factor in lipoprotein metabolism and atherogenesis. Here, we report that chronic high-fat, high-cholesterol diet feeding markedly increased plasma cholesterol levels in C57BL/6 mice. PLTP deficiency attenuated diet-induced hypercholesterolemia by dramatically reducing apolipoprotein E-rich lipoproteins (-88%) and, to a lesser extent, LDL (-40%) and HDL (-35%). Increased biliary cholesterol secretion, indicated by increased hepatic ABCG5/ABCG8 gene expression, and decreased intestinal cholesterol absorption may contribute to the lower plasma cholesterol in PLTP-deficient mice. The expression of proinflammatory genes (intercellular adhesion molecule-1 and vascular cell adhesion molecule-1) is reduced in aorta of PLTP knockout mice compared with wild-type mice fed either a chow or a high-cholesterol diet. Furthermore, plasma interleukin-6 levels are significantly lower in PLTP-deficient mice, indicating reduced systemic inflammation. These data suggest that PLTP appears to play a proatherogenic role in diet-induced hyperlipidemic mice.

Atherogenic, enlarged, and dysfunctional HDL in human PLTP/apoA-I double transgenic mice

In low density lipoprotein receptor (LDLR)-deficient mice, overexpression of human plasma phospholipid transfer protein (PLTP) results in increased atherosclerosis. PLTP strongly decreases HDL levels and might alter the antiatherogenic properties of HDL particles. To study the potential interaction between human PLTP and apolipoprotein A-I (apoA-I), double transgenic animals (hPLTPtg/hApoAItg) were compared with hApoAItg mice. PLTP activity was increased 4.5-fold. Plasma total cholesterol and phospholipid were decreased. Average HDL size (analyzed by gel filtration) increased strongly, hPLTPtg/hApoAItg mice having very large, LDL-sized, HDL particles. Also, after density gradient ultracentrifugation, a substantial part of the apoA-I-containing lipoproteins in hPLTPtg/hApoAItg mice was found in the LDL density range. In cholesterol efflux studies from macrophages, HDL isolated from hPLTPtg/hApoAItg mice was less efficient than HDL isolated from hApoAItg mice. Furthermore, it was found that the largest subfraction of the HDL particles present in hPLTPtg/hApoAItg mice was markedly inferior as a cholesterol acceptor, as no labeled cholesterol was transferred to this fraction. In an LDLR-deficient background, the human PLTP-expressing mouse line showed a 2.2-fold increased atherosclerotic lesion area. These data demonstrate that the action of human PLTP in the presence of human apoA-I results in the formation of a dysfunctional HDL subfraction, which is less efficient in the uptake of cholesterol from cholesterol-laden macrophages.

Lipoprotein inflammatory properties and serum amyloid A levels but not cholesterol levels predict lesion area in cholesterol-fed rabbits

Rabbits on a 1% cholesterol diet received injections of vehicle with or without D-4F or L-4F. After 1 month, the percent of aorta with atherosclerotic lesions was 24 +/- 15% (vehicle), 10 +/- 6% (D-4F) (P < 0.01 vs. vehicle), and 13 +/- 9% (L-4F) (P < 0.05 vs. vehicle). Inflammatory indexes for HDL and LDL were determined by measuring monocyte chemotactic activity after adding rabbit lipoproteins to human endothelial cells. HDL-inflammatory index (HII) and LDL-inflammatory index (LII), respectively, were 1.39 +/- 0.24; 1.35 +/- 0.29 (vehicle), 0.67 +/- 0.26; 0.63 +/- 0.38 (D-4F) (P < 0.001 vs. vehicle), and 0.67 +/- 0.2; 0.68 +/- 0.32 (L-4F) (P < 0.01 vs. vehicle). Serum amyloid A (SAA) levels were 95 +/- 39, 8 +/- 22, and 7 +/- 19 mug/ml, respectively, for vehicle, D-4F, and L-4F (P < 0.001 vs. vehicle). There was no correlation between lesion area and total plasma or HDL-cholesterol levels. In contrast, there was a positive correlation with HII, LII, and SAA (P = 0.002, P = 0.0026, P = 0.0079, respectively). HII correlated closely with SAA levels (r = 0.6616; r(2) = 0.4377, P < 0.0001). Thus, HII, LII, and SAA are better predictors of lesion area than are total plasma or HDL-cholesterol levels in cholesterol-fed rabbits.

Insoluble Aggregates and Protease-resistant Conformers of Prion Protein in Uninfected Human Brains

Aggregated prion protein (PrPSc), which is detergent-insoluble and partially proteinase K (PK)-resistant, constitutes the major component of infectious prions that cause a group of transmissible spongiform encephalopathies in animals and humans. PrPSc derives from a detergent-soluble and PK-sensitive cellular prion protein (PrPC) through an alpha-helix to beta-sheet transition. This transition confers on the PrPSc molecule unique physicochemical and biological properties, including insolubility in nondenaturing detergents, an enhanced tendency to form aggregates, resistance to PK digestion, and infectivity, which together are regarded as the basis for distinguishing PrPSc from PrPC. Here we demonstrate, using sedimentation and size exclusion chromatography, that small amounts of detergent-insoluble PrP aggregates are present in uninfected human brains. Moreover, PK-resistant PrP core fragments are detectable following PK treatment. This is the first study that provides experimental evidence supporting the hypothesis that there might be silent prions lying dormant in normal human brains.

Anti‐tumour necrosis factor‐α activity in Ixodes ricinus saliva

Tumour necrosis factor-alpha (TNF-alpha) is one of the most prominent inflammatory mediators playing a central role in starting off the inflammatory reactions of the innate immune system. We identified a TNF-alpha-inhibitory activity in the saliva and salivary gland extract (SGE) from partially fed Ixodes ricinus ticks. Using mouse and human TNF-alpha specific ELISA, we showed that tick saliva or SGE markedly reduced the level of detectable cytokine. Both saliva and SGE inhibited the cytotoxic effect of TNF-alpha in a bioassay. Elimination of the TNF-alpha-inhibitory activity in SGE by trypsin digestion demonstrated that the anti-TNF-alpha factor is a protein. Fast protein liquid chromatography fractionation of SGE showed one peak of TNF-alpha-inhibitory activity corresponding to a protein with estimated molecular mass 23 kDa. The likely mechanism of the inhibitory effect is a direct binding of the cytokine. The TNF-alpha-inhibitory molecule seems to play an important role in the anti-inflammatory effect of tick saliva at the tick feeding site, providing a gateway to the host for tick-borne pathogens.

Identification of peptides in antimicrobial fractions of cheese extracts by electrospray ionization ion trap mass spectrometry coupled to a two‐dimensional liquid chromatographic separation

Electrospray ionization ion trap mass spectrometry (ESI-ITMS) coupled to a two-dimensional liquid chromatographic separation was applied to the identification of peptides in antimicrobial fractions of the aqueous extracts of nine Italian cheese varieties. In particular, the chromatographic fractions collected during a preliminary fast protein liquid chromatography (FPLC) separation on the cheese extracts were assayed for antimicrobial activity towards Lactobacillus sakei A15. Active fractions were subsequently analyzed by reversed-phase high-performance liquid chromatography electrospray ionization sequential mass spectrometry (HPLC/ESI)-ITMSn, with n up to 3. Peptide identification was then performed starting from a conventional proteomics approach based on tandem mass spectrometric (MS/MS) analysis followed by database searching. In many cases this strategy had to be integrated by a careful correlation between spectral information and predicted peptide fragmentation, in order to reach unambiguous identifications. When even this integrated approach failed, MS3 measurements provided decisive information on the amino acid sequence of some peptides, through fragmentation of pendant groups along the peptide chain. As a result, 45 peptides, all arising from hydrolysis of milk caseins, were identified in nine antimicrobial FPLC fractions of aqueous extracts obtained from five of the nine cheese varieties considered. Many of them corresponded to peptides already known to exhibit biological activity.

Identification of Ghrelin in Human Saliva: Production by the Salivary Glands and Potential Role in Proliferation of Oral Keratinocytes

We investigated whether ghrelin is present in human saliva, is produced by salivary glands, and physiologic consequences of these findings. Expression of ghrelin and specific receptor mRNA was determined by PCR. Proteins were identified by immunoblotting and size-exclusion fast protein liquid chromatography (FPLC) with consecutive RIA. Specific RIAs were used for quantification of salivary total and bioactive ghrelin. Distribution of ghrelin was investigated by immunohistochemistry in cryosections of the salivary glands. The effect of ghrelin on incorporation of 5-bromo-2'-deoxyuridine as a measure of cell proliferation was investigated in primary oral keratinocytes. Ghrelin is produced by the salivary glands. The hormone was identified in saliva and glands by immunoblotting and by FPLC fractionation of saliva. Immunohistochemistry demonstrated ghrelin distribution in the salivary glands. The receptor was also produced by the glands and by oral keratinocytes and was shown to be functional. Comparison of total ghrelin values for healthy individuals (body mass index, 18-27 kg/m2) showed significantly lower concentrations in saliva than in serum (P < 0.01). The correlation between both matrices was r2 = 0.56 (P < 0.001) with a negative correlation to body mass index (r2 = 0.314; P < 0.01). Bioactive acylated ghrelin was also present in saliva. Incubation of keratinocytes with ghrelin led to significantly increased cell proliferation (P < 0.001). This effect could be completely suppressed by co-incubation with NOX-B11 (50 nmol/L), a novel specific inhibitor of acylated ghrelin. Ghrelin in saliva is produced and released by salivary glands. The effect of ghrelin on oral cell proliferation adds to the pro-proliferative action of other salivary growth factors.

A Novel in Vivo Lecithin-Cholesterol Acyltransferase (LCAT)-Deficient Mouse Expressing Predominantly LpX Is Associated with Spontaneous Glomerulopathy

Complete lecithin cholesterol acyltransferase (LCAT) deficiency is a rare genetic cause of extreme reduction in high density lipoproteins and there is a high prevalence of chronic renal dysfunction that may progress to renal failure. Previous in vitro studies suggest the vesicular lipoprotein X (LpX) particles commonly seen in LCAT-deficient plasmas may be causative. To test this hypothesis, we have generated a novel murine model that selectively accumulate LpX in the circulation by cross breeding the sterol regulatory element binding protein (SREBP) 1a transgenic mice (S+) with the LCAT knockout (lcat-/-) mice. Fast protein liquid chromatography fractionation of pooled plasma lipids revealed that virtually all cholesterol is concentrated in the very low density lipoprotein (VLDL)-sized fractions. These fractions are enriched in free cholesterol and phospholipid but extremely poor in triglyceride. Electron microscopy of the d <1.063 g/ml fraction of the S+lcat-/- mice revealed abnormal large vesicular particles, suggestive of LpX. The S+lcat-/- mice developed glomerular lesions spontaneously evident at 6 months with glomerular and tubulointerstitial lipid-deposits. Immunohistochemical staining with RhoA showed marked positive focal staining in glomeruli in the S+lcat-/- mice and undetectable in the S+/lcat+/+ control. By 10 months of age, the kidneys showed progressive glomerular injury including segmental foam cell infiltrates, mesangial expansion, and hyalinosis. Renal abnormalities are very similar to those seen in human LCAT deficiency. We conclude that the selective high-level accumulation of plasma LpX in the S+lcat-/- mice is strongly associated with a spontaneous glomerulopathy, providing in vivo evidence that LpX contributes to the LCAT deficiency-related nephropathy.

Echinococcus granulosus coproantigens: chromatographic fractionation and characterization.

Dogs infected with adult tapeworms of Echinococcus granulosus release antigens (coproantigens) in faeces which can be detected by a capture ELISA. Supernatants prepared from E. granulosus-infected dog faecal samples were fractionated by size-exclusion fast protein liquid chromatography (FPLC) on a Superose-6 column. Coproantigen ELISA and Western blotting were used to demonstrate the immunoreactivity of eluted fractions. Two main FPLC peaks of antigenic activity were detected and designated as fraction F1 and fraction F2 with approximate relative molecular weights > 670 kDa, and in the range of 146 to 440 kDa respectively. These two antigenic fractions (F1 and F2) fractionated from infected dog faeces were heat stable and largely protease-insensitive, but were highly sensitive to sodium periodate treatment, which strongly suggested the involvement of carbohydrates. Capture IgG antibodies against E. granulosus proglottis somatic extracts, detected a molecule with an approximate molecular weight of 155 kDa in fraction F2 after immunoblotting. The 155 kDa antigen could be completely ablated by sodium periodate treatment, but not after protease or lipase treatment. A surface tegument preparation of adult E. granulosus tapeworms contained large amounts of antigen that corresponded in size range and antigenicity to that observed in the FPLC fraction F2. There was also a peak of antigenic activity at > 670 kDa corresponding to fraction F1 from a culture derived excretory-secretory (E-S) adult tapeworm preparation. The involvement of carbohydrate moieties in coproantigen activity present in the FPLC fractions F1 and F2 from faecal supernatants of E. granulosus-infected dogs was confirmed by lectin-binding assays and exoglycosidase treatment, which showed that alpha-D-mannose and/or alpha-D-glucose, beta-galactose and N-acetyl-beta-glucosamine residues were the most important carbohydrate components in putative coproantigens present in both fractions. N-acetyl-beta-glucosamine and sialic acid residues were also contained in coproantigen molecules present in fraction F2. These results suggested that coproantigens detected in faeces of E. granulosus-infected dogs are large molecular weight molecules that may be derived from the carbohydrate-rich surface glycocalyx of adult worms, and are shed, released or secreted during the life-span of the tapeworm.

Effect of fast protein liquid chromatography fractionated salivary gland extracts from different ixodid tick species on interleukin-8 binding to its cell receptors

Interleukin-8 plays a critical role in inflammatory processes. Hence generation of molecules with anti-IL-8 activity is likely to be important for successful feeding and for survival of the ticks. Anti-IL-8 activity was studied in saliva of three ixodid tick species--Dermacentor reticulatus (Fabricius, 1794), Rhipicephalus appendiculatus Neumann, 1901, and Amblyomma variegatum (Fabricius, 1794). The greatest activity was shown in saliva prepared from D. reticulatus. The activity was attributed to tick salivary gland molecules that bind to IL-8, preventing binding of the chemokine to its specific receptor, rather than to occupation of the IL-8 cell receptor by the tick molecules. The distribution of anti-IL-8 activity in fast protein liquid chromatography (FPLC) fractions of salivary gland extracts (SGE) derived from adult female D. reticulatus, R. appendiculatus and A. variegatum was compared directly by both ELISA and receptor-binding inhibition assays. The correspondence in results with fractions of SGE from ELISA is consistent with detection of tick molecules that inhibit IL-8 binding to its receptor. As IL-8 is an important chemoattractant and activator of neutrophils, the presence of an anti-IL-8 activity in tick saliva indicates that neutrophils play an important role in the host response to parasitism by ticks.

Novel therapeutic strategies for leiomyomas: targeting growth factors and their receptors.

Leiomyomas (fibroids) are benign smooth-muscle cell (SMC) tumors of the uterus and are the most common pelvic tumors in women. These tumors occur primarily during the reproductive years and are the most common indication for hysterectomy in women. Unfortunately the only effective treatments for leiomyomas and the associated abnormal uterine bleeding are surgical, involving either hysterectomy, myomectomy, or hysteroscopic removal of the tumors. The goal of this paper is to discuss recent research findings that support the idea of using therapeutic compounds that block the actions of specific growth factors as therapeutic agents for treatment of leiomyomas and abnormal uterine bleeding. Most of the studies were carried out using cell cultures of leiomyoma or myometrial SMCs. Primary cultures of SMCs provide a system for investigation of the roles of growth factors and their receptors in proliferation of normal myometrial and leiomyoma SMCs. Several growth factors have been shown to be present and to have regulatory roles in the proliferation of uterine SMCs. Bioassay and Western blotting of fast protein liquid chromatography fractions of tissue extracts identified platelet-derived growth factor, heparin-binding epidermal growth factor, hepatoma-derived growth factor, and basic fibroblast growth factor in normal myometrium and fibroid tumors. The presence of heparin-binding growth factors suggests a possible focus for therapeutic agents. RG13577 (a heparinlike compound) and halofuginone (an alkyloid) reversibly inhibited DNA synthesis of normal myometrial and leiomyoma cells without toxic effects. Pirfenidone, a known antifibrotic drug, inhibited DNA synthesis and synthesis of collagen type I mRNA in normal and fibroid cells, and decreased collagen type III mRNA only in normal myometrial cells. Another hopeful therapeutic candidate, interferon-Alpha, significantly inhibited growth factor-stimulated proliferation in both normal and leiomyoma cells. These results suggest that future nonsurgical treatments for leiomyomas may include compounds that block the actions of specific growth factors that regulate proliferation and collagen production by uterine SMCs.