Purification and characterization of angiogenic factor from human bladder cancer

An angiogenic factor from human transitional cell cancer of bladder was purified by protein extraction, cation exchange chromatography, gel filtration high-performance liquid chromatography (GE-HPLC) and reversed-phase high-performance liquid chromatography (RP-HPLC). The purified substance was named as bladder cancer angiogenic factor (BCAF). Biological activity of the BCAF was assessed by using the method of chick embryo chorioallantoic membrane (CAM) assay and 3H-TdR incorporation into DNA in Balb/c 3T3 cells. The BCAF displayed the potent activities of neovascularization in CAM and DNA synthesis in Balb/c 3T3 cells. The ultrastructural features of blood vessels induced by the BCAF were similar to the blood vessels in tumors. The BCAF contained a protein with an approximate molecular weight of 15,000 D, which was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining. Amino acid compositions of the BCAF were also analysed by acid hydrolysis.     

The role of neutrophils and platelet-activating factor in mediating experimental pancreatitis

BACKGROUND & AIMS: Pancreatitis is characterized by inflammation and death of acinar cells. Death can occur by either necrosis or apoptosis. The initial injury may cause expression of cytokines that mediate activation and infiltration of neutrophils. The aim of this study was to assess the effect of neutrophils and platelet-activating factor (PAF) in cell death responses. METHODS: The effects of neutrophil depletion with antineutrophil serum (ANS) and a PAF antagonist (BN52021) were measured in the cerulein model of pancreatitis. Rats received a 6-hour intravenous infusion of cerulein either alone or after treatment with ANS, BN52021, or both. RESULTS: Cerulein-induced pancreatitis was characterized by neutrophilic infiltration, vacuolization of acinar cells, and foci of necrosis. Treatment with ANS and BN52021 prevented the inflammatory response caused by cerulein and decreased the cell damage. Treatment with ANS increased apoptosis in cerulein-infused animals. When BN52021 was added, apoptosis was abolished. The measurement of PAF in pancreatic tissue showed a ninefold increase with cerulein treatment alone and a 14-fold increase in cerulein-infused, neutrophil-depleted animals. CONCLUSIONS: The results indicate that cerulein stimulates pancreatic production of PAF. PAF mediates both apoptosis and neutrophil chemotaxis in the pancreas. Neutrophils in turn may convert acinar cells undergoing apoptosis into necrotic cells.     

Regulation of leukotriene and platelet-activating factor synthesis in human alveolar macrophages

It has been suggested that phospholipase A2 (PLA2) contributes to the regulation of leukotriene (LT) and platelet-activating factor (PAF) synthesis by controlling the release of their precursors, arachidonic acid (AA) and lysophosphatidylcholine (lysoPC), from membrane phospholipids. In rat alveolar macrophages (AMs), PLA2 appears to have a major role in LT synthesis but a more limited role in PAF synthesis. The present study was designed to define the role of PLA2 in LT and PAF synthesis in human AMs and determine whether differences exist between AMs obtained from normal subjects and those from patients with asthma. In the normal subjects, the calcium ionophore A23187 (Cal) increased AM PAF synthesis (percent incorporation of tritiated acetate) by 135% (p < 0.01) and LTB4 synthesis 88-fold (p < 0.001). Phorbol myristate acetate (PMA) had little effect alone, but it had a synergistic effect with Cal, increasing PAF synthesis by 466% and LTB4 synthesis to 229-fold above the control values (p < 0.001 for both). Ro 25-4331, a combined cytosolic (c) and secretory (s) PLA2 inhibitor, had little effect on the Cal-stimulated PAF synthesis, but it completely blocked the effect of PMA. It also blocked the Cal- and Cal+PMA-stimulated LTB4 synthesis. AACOCF3, a cPLA2 inhibitor, had no effect on either Cal or Cal+PMA-stimulated PAF synthesis. It reduced LTB4 synthesis, but it did so less effectively than Ro 25-4331. CoA-independent transacylase (CoAI-TA) activity in the AMs increased after stimulation and exposure to Ro 25-4331. SK&F 45905, a CoAI-TA inhibitor, reduced stimulated PAF synthesis by 30% to 40%. Patients with asthma had similar results except that cPLA2 had a greater role in stimulated LTB4 synthesis. These data indicate that PLA2 plays a direct role in human AM LT synthesis; both the cytosolic and secretory forms contribute to LT synthesis; PLA2 appears to have a more limited role in PAF synthesis, although it mediates the synergistic effect of PMA, probably via sPLA2; and CoAI-TA contributes to PAF synthesis during PLA2 inhibition. With the exception of the greater role for cPLA2 in stimulated LTB4 synthesis in the patients with asthma, the contributions of PLA2 and CoAI-TA to AM LT and PAF synthesis appear to be similar in normal subjects and patients with asthma.     

The role of endogenous nitric oxide and platelet-activating factor in hypoxia-induced intestinal injury in rats

BACKGROUND/AIMS: Nitric oxide is an endothelium-derived relaxing factor that promotes capillary integrity, inhibits leukocyte adherence and activation, and scavenges oxygen radicals. Because these effects are important in experimental intestinal injury, we studied the role of NO inhibition on hypoxia-induced bowel necrosis in the rat and investigated the interaction between platelet-activating factor (PAF) and NO in this model. METHODS: Sprague-Dawley rats were treated with either hypoxia, NO synthase inhibition (NG-methyl-L-arginine [LNMA] or NG-nitro-L-arginine methyl ester [L-NAME]), hypoxia+LNMA, hypoxia+LNMA+NO donors, or hypoxia+LNMA+PAF receptor inhibition. Evaluations included blood pressure, superior mesenteric artery blood flow, arterial blood gases, histological intestinal injury, intestinal myeloperoxidase activity, and intestinal PAF activity. RESULTS: We found that hypoxia alone for 90 minutes (10% O2, partial O2 pressure = 45 mm Hg) or LNMA alone had no detrimental effects. However, hypoxia+LNMA together caused hypotension, metabolic acidosis, intestinal injury, increased intestinal myeloperoxidase activity, and elevated intestinal PAF concentrations that were prevented by exogenous L-arginine. Furthermore, the hypotension and intestinal injury was prevented by PAF receptor blockade. CONCLUSIONS: We conclude that endogenous NO protects the intestine from hypoxia-induced inflammation and injury, and the balance between local PAF and NO modulates the outcome of hypoxia-stressed intestine.     

The role of recombinant platelet-activating factor acetylhydrolase in a neonatal rat model of necrotizing enterocolitis

Previous studies have shown that the endogenous inflammatory mediator platelet-activating factor (PAF) plays an important role in the pathophysiology of neonatal necrotizing enterocolitis (NEC). This study was designed to investigate the role of the PAF-degrading enzyme acetylhydrolase (PAF-AH) in a neonatal rat model of NEC. To study the absorption, localization, and activity of human recombinant PAF-AH (rPAF-AH), newborn rats were treated with enteral rPAF-AH, and plasma and intestines were sampled at 8 and 24 h for determination of PAF-AH enzyme activity and rPAF-AH concentration using a specific enzyme-linked immunoassay. To study the effect of rPAF-AH on neonatal NEC, rats were treated with rPAF-AH via the enteral route every 3 h, and then subjected to formula feeding and asphyxia per an established neonatal rat protocol for NEC. Pretreatment with enteral rPAF-AH significantly reduced the incidence of NEC compared with controls (6/26 versus 19/26, p < 0.001). We found that enteral rPAF-AH administration resulted in significant intestinal PAF-AH activity but no circulating PAF-AH activity despite immunohistochemical localization of the administered rPAF-AH to the intestinal epithelial cells. These findings suggest that rPAF-AH is functional and stable in the gut of neonatal rats. We conclude that enteral administration of rPAF-AH remains locally active and reduces the incidence of NEC in our experimental animal model.     

Basic fibroblast growth factor receptors and their prognostic value in human breast cancer

We performed a saturation binding study with 125I-labeled FGF (fibroblast growth factor)-2 in a nonselected series of 250 human primary breast cancers. Two hundred twenty-five breast cancer biopsies possessed bFGFR (basic FGF receptor). The median dissociation constant was 0.35 nM (range, 0.014-1.9), and the median concentration was 1126 fmol/mg protein (range, 49-7328). FGFR-1 was localized, using a specific monoclonal antibody, in cancerous cells and in epithelial cells in normal breast or in benign tumors. In all of the tissues studied, light stromal cell staining was also observed. Thus, the localization of FGFR-1 in carcinoma cells supports the hypothesis that an important part of FGF-2 binding reflects binding to FGFR-1. bFGFR concentrations were positively correlated to estrogen receptor and progesterone receptor levels. Cox univariate analyses showed that the bFGFR (> or = upper quartile) was associated to longer relapse-free survival [P = 0.004; RR (risk ratio), 0.46] and overall survival (P = 0.001; RR, 0.35); age, estrogen receptor levels, progesterone receptor levels, node involvement, tumor diameter, and histoprognostic grading were prognostic, also. In Cox multivariate analyses, only the bFGFR, age, node involvement, and histoprognostic grading were prognostic factors; the bFGFR was associated with longer relapse-free survival (P = 0.03; RR, 0.4) and overall survival (P = 0.009; RR, 0.3). The present study confirms that FGF could be an important regulator of human breast cancer growth and that patients with a high level of bFGFR had a better prognosis.     

Sensitive densitometry for the determination of platelet-activating factor and other phospholipids in human tears

A sensitive TLC method is reported for the determination of the platelet-activating factor (PAF) and other phospholipids in human tears. Tear samples absorbed on filter-paper were subjected to diatomite column extraction with chloroform-methanol. The eluent containing phospholipids was spotted on a silica gel plate. After removing lipids other than phospholipids by pre-development with hexane-diethyl ether (4 + 1 v/v), individual phospholipid separation was carried out by development with chloroform-methanol-water (65 + 35 + 7 v/v). Phospholipase C and then alkaline phosphatase solutions were sprayed on the TLC plate at 45 degrees C to liberate phosphate from each phospholipid. By spray application of a mixture of ammonium molybdate and Malachite Green, the liberated phosphates appeared as blue-green spots of molybdophosphate-Malachite Green aggregate on a yellow-brown background. The absorbance of each spot on the plate was measured at 620 nm with a densitometer. The peak area was found to be linearly related to PAF content in the range 2-100 pmol per spot, the RSD being 2% (n = 7). Approximately 86% of standard PAF added to tears was recovered. By this method, lysophosphatidylcholine (lysoPC), phosphatidylcholine (PC) and phosphatidylethanolamine (PE) in human tears could also be detected with high sensitivity. Each phospholipid in healthy human tears showed a nearly constant content; PAF, lysoPC, PC and PE were present at levels of 26.2, 42.3, 10.0 and 19.7%, respectively.     

Inhalation of platelet-activating factor induced guinea pig airway hyperresponsiveness: role of eosinophils activation

Exposure of guinea pigs to aerosols of 200 micrograms/ml platelet-activating factor 24 h later, airway hyperresponsiveness to histamine induced and number of eosinophils and hypodense eosinophils in bronchoalveolar lavage fluid (BALF) increased, comparing with the control group. The number of eosinophils in BALF was corelated with PC20 value in PAF-treated group (r = -0.62, P < 0.05). However, the percentage of hypodense eosinophil in BALF had closer relation to airway responsiveness (r = -0.84, P < 0.01). The content of peroxidase in hypodense eosinophils in BALF for guinea pigs treated by inhalation of PAF was lowered markedly than that in normodense eosinophil (P < 0.05). The result suggested that chemotaxis and activation of eosinophils by PAF might play an important role in airway hyperresponsiveness.     

Endocrine response and resistance in breast cancer: a role for the transcription factor Fos

The concentration of the tumor marker CA 19-9 is influenced by the patient's secretor status and Lewis genotype. The aim of this study was to establish novel reference intervals for CA 19-9 in serum based on secretor and Lewis genotypes, to investigate the biological variation of CA 19-9, and to evaluate the utility of Lewis and secretor genotyping on a group of individuals with serologically defined Lewis phenotypes. CA 19-9 was measured in serum of 500 healthy individuals. Secretor and Lewis genotypes were determined by sequencing and PCR-cleavage methods. Significant differences were found between subgroups with different Lewis and secretor genotypes. Genotype-based reference intervals for CA 19-9 are presented. The upper reference limit for all individuals was 28.7 kilounits/L; for secretors and nonsecretors, the upper reference limits were 12.4 and 61.2 kilounits/L, respectively. The analytical imprecision (CVA) was 9.8%, the within-subject variability (CVI) was 15.8%, and the between-subject variability (CVG) was 102.2%. Good agreement was found between Lewis and secretor genotyping and conventional blood grouping. Genotype-based reference intervals may be a way to increase the clinical utility of CA 19-9. On the basis of the calculation of a critical difference for sequential values (significant at P 相似文献   

The role of N-glycosylation for functional expression of the human platelet-activating factor receptor. Glycosylation is required for efficient membrane trafficking

Streptococcus pneumoniae has been shown to utilize the platelet activating factor receptor for binding and invasion of host cells (Cundell, D. R., Gerard, N. P., Gerard, C., Idanpaan-Heikkila, I., and Tuomanen, E. I. (1995) Nature, in press). Because bacterial binding is in part carbohydrate dependent, and the human platelet-activating factor (PAF) receptor bears a single N-linked glycosylation sequence in the second extracellular loop, we undertook studies to determine the role of this epitope in PAF receptor function. Binding of pneumococci to COS cells transfected with the human PAF receptor is greatly reduced for a receptor mutant that bears no N-linked glycosylation site. Immunohistochemical and binding analyses show decreased expression of the non-glycosylated molecule on the cell membrane relative to the wild type receptor; however, metabolic labeling and immunopurification indicate it is synthesized intracellularly at a level similar to the native molecule. A mutant receptor encoding a functional glycosylation site at the NH2 terminus is better expressed at the cell surface compared with the non-glycosylated form, indicating that trafficking to the cell surface is facilitated by glycosylation, but its location is relatively unimportant. The binding affinity for PAF is not significantly effected by the presence or location of the carbohydrate, and variations in cell surface expression have little influence on signal transduction, as the non-glycosylated PAF receptor is equally effective for activation of phospholipase C as the native molecule. These data are supportive of pneumococcal binding on protein moiety(ies) of the PAF receptor and indicate that N-glycosylation facilitates expression of the protein on the cell membrane.     

Hepatocyte growth factor in human breast milk

BACKGROUND: Glomerular accumulation of macrophages/monocytes (M/M) is a typical early feature in the course of anti-thymocyte serum (ATS)-induced nephritis. We have previously shown that glomerular synthesis and expression of monocyte-chemoattractant protein-1 (MCP-1) occurs before influx of M/M and a neutralizing anti-MCP-1 antibody reduced this cell infiltrate by one third. The present study was undertaken to test the effect of two angiotensin II type 1 (AT1) receptor antagonists, losartan and irbesartan, on ATS-stimulated MCP-1 expression as well as glomerular influx of M/M. METHODS: Treatment of rats with either losartan or irbesartan was started 24 h before administration of ATS. After 24 h, MCP-1 mRNA expression was evaluated by RT-PCR and Northern blots. MCP-1 protein was determined by Western blots and chemotactic factors released from isolated glomeruli were measured by chemotactic assay. Kidney sections were stained for rabbit IgG, complement C3, and M/M (ED1 antigen). RESULTS: Both AT1-receptor antagonists caused a significant, but not total reduction in MCP-1 mRNA and protein expression 24 h after injection of ATS. Treatment with losartan or irbesartan also reduced the chemotactic activity of isolated glomeruli from nephritic animals. Quantification of ED1-positive cells revealed that losartan as well as irbesartan reduced glomerular M/M invagination in nephritic rats by approximately 30-50%. However, treatment with AT1-receptor antagonists did not influence binding of ATS to mesangial cells and subsequent complement activation indicating that the attenuated MCP-1 expression is not due to differences in delivery and binding of ATS to mesangial cells. CONCLUSION: Our data indicate that short-term antagonism of AT1 receptors abolished the early glomerular MCP-1 expression and M/M influx. These results indicate that angiotensin II may exert immunomodulatory effects in vivo and adds a new mechanism showing how this vasopeptide may be involved in the pathogenesis of renal diseases.     

Comparison of radioligand assay and immunostaining for epidermal growth factor receptor in human breast cancer

Epidermal growth factor (EGF) receptor status is a useful prognostic indicator in women with breast cancer. Lack of standardization and correlation of methodology for the detection of EGF receptor has hampered its further evaluation. EGF receptor status was ascertained by immunohistochemistry and radioligand assay in 120 breast cancers. Of 52 tumours negative for EGF receptor on radioligand assay, 47 were negative on immunohistochemistry and, of 68 tumours positive for the receptor on assay, 52 were positive on immunohistochemistry. If the more widely evaluated radioligand assay is assumed to be the 'gold standard', immunohistochemistry has a sensitivity of 81 per cent and a specificity of 91 per cent.     

Metabolism of platelet-activating factor in rat epididymal spermatozoa

A role for platelet-activating factor (PAF) in sperm function has been proposed. In the present investigation the metabolism of PAF was examined in sperm and epididymal tissue. The major regulatory enzymes for the synthesis of PAF via the de novo pathway have been established in spermatozoa; these include acetyltransferase and cholinephosphotransferase specific for PAF biosynthesis. De novo acetyltransferase activity for PAF biosynthesis in spermatozoa was significantly higher than that of the acetyltransferase of the remodeling pathway. A metabolic pathway was described for the catabolism of PAF in sperm, involving PAF-acetylhydrolase (-AH), lysophospholipase D, and a phosphohydrolase. An isozyme of PAF-AH similar to that reported in sera was also demonstrated in epididymal fluid and tissue. This isozyme was distinctly different from that found in the spermatozoa. The partial inactivation of PAF-AH by the vaginal pH, and/or its detachment from sperm during migration to the site of fertilization, may allow increased motility and migration to the site of fertilization. It is suggested that a decapacitation factor previously described may be related to PAF-AH.     

Generation of reactive oxygen species and activity of platelet-activating factor acetylhydrolase in human monocyte-derived macrophages

Monocytes were prepared from healthy human volunteers and were allowed to differentiate into macrophages by adhesion to plastic surface and cultured over 7 days in presence of either 10% fetal calf serum (FCS), human control serum or serum from hyperlipaemic patients. Hyperlipaemic serum stimulated the differentiation (measured as an increase in cellular protein and DNA content) to a higher extent when compared to control serum and FCS. With all sera a marked increase of the cellular activity of the enzyme platelet-activating factor acetylhydrolase (PAF-AH) and a tremendous decrease in the capacity of cells to generate reactive oxygen species (ROS) was observed. After seven days of culture the increase in PAH-AH activity was about 19-fold with hyperlipaemic serum, 11-fold with control serum and 6-fold with FCS. During the same period of time ROS generation measured as zymosan-induced chemiluminescence decreased by about 98% and no significant differences between the three types of serum were found. The results indicate that the activity of PAF-AH and the capacity of ROS generation which are both assumed to play an important role in the oxidation of low-density lipoproteins (LDL) and thus in the development of atherosclerosis, change in opposite direction during the differentiation of blood monocytes into macrophages, and that hyperlipaemic serum stimulates PAF-AH activity but not ROS generation.