Molecular cloning and characterization of a radial spoke head protein of sea urchin sperm axonemes: involvement of the protein in the regulation of sperm motility.

Monoclonal antibodies raised against axonemal proteins of sea urchin spermatozoa have been used to study regulatory mechanisms involved in flagellar motility. Here, we report that one of these antibodies, monoclonal antibody D-316, has an unusual perturbating effect on the motility of sea urchin sperm models; it does not affect the beat frequency, the amplitude of beating or the percentage of motile sperm models, but instead promotes a marked transformation of the flagellar beating pattern which changes from a two-dimensional to a three-dimensional type of movement. On immunoblots of axonemal proteins separated by SDS-PAGE, D-316 recognized a single polypeptide of 90 kDa. This protein was purified following its extraction by exposure of axonemes to a brief heat treatment at 40 degrees C. The protein copurified and coimmunoprecipitated with proteins of 43 and 34 kDa, suggesting that it exists as a complex in its native form. Using D-316 as a probe, a full-length cDNA clone encoding the 90-kDa protein was obtained from a sea urchin cDNA library. The sequence predicts a highly acidic (pI = 4.0) protein of 552 amino acids with a mass of 62,720 Da (p63). Comparison with protein sequences in databases indicated that the protein is related to radial spoke proteins 4 and 6 (RSP4 and RSP6) of Chlamydomonas reinhardtii, which share 37% and 25% similarity, respectively, with p63. However, the sea urchin protein possesses structural features distinct from RSP4 and RSP6, such as the presence of three major acidic stretches which contains 25, 17, and 12 aspartate and glutamate residues of 34-, 22-, and 14-amino acid long stretches, respectively, that are predicted to form alpha-helical coiled-coil secondary structures. These results suggest a major role for p63 in the maintenance of a planar form of sperm flagellar beating and provide new tools to study the function of radial spoke heads in more evolved species.

Cytosolic proteins of 21-23 kDa are methylated by kidney cortex membrane-associated C-terminal carboxyl methyltransferases.

We have studied the effect of a soluble fraction from kidney cortex on the C-terminal carboxyl methylation of 21-23 kDa proteins catalyzed by membrane-associated methyltransferases. Addition of soluble proteins to isolated luminal, antiluminal and intracellular membranes resulted in a large increase in the methylation of the membrane-associated 21-23 kDa substrates. Fractionation of the soluble extract from the cortex by Q-Sepharose anion exchange chromatography showed the presence of two distinct peaks of proteins presenting stimulating activities, eluting at 0.15 M (peak I) and 0.4 M (peak II) NaCl, respectively. Both peaks eluted as proteins of apparent molecular sizes of 40 kDa upon Superose 6 gel-filtration chromatography. No methylation activity towards N-acetyl-S-trans,trans-farnesylcysteine (AFC), a good substrate for C-terminal carboxyl methyltransferases, was associated with either peaks. In contrast, the increase in methylation induced by these proteins was strongly inhibited by AFC, suggesting that the methylation induced by these factors occurred on C-terminal isoprenylated cysteine residues. Both partially purified proteins competitively inhibited the methylation of AFC by the membrane-associated enzymes, suggesting that they may represent substrates for the methyltransferases. Immunoblotting of these partially purified soluble substrates with a rabbit polyclonal antibody directed against the small G-protein CDC42 showed the presence of this protein in peak I but not in peak II. Taken together, these results suggest the presence in a soluble fraction from the kidney of distinct methyl-accepting proteins, one of these being tentatively identified as the small G-protein CDC42.

Guanine nucleotides stimulate carboxyl methylation of kidney cytosolic proteins.

We studied the effect of guanine nucleotides on the carboxyl methylation catalyzed by class II protein carboxylmethyltransferases (PCMT). Addition of guanosine 5'-O-(gamma-thio)triphosphate (GTP gamma S) promoted a time- and concentration-dependent enhancement of protein methylation in the cytosolic fraction isolated from kidney cortex. GTP gamma S affected the kinetics of the methylation reaction, as reflected by alterations of both apparent Km and Vmax of the methyltransferase. This effect was specific for guanine nucleotides and was completely abolished by addition of S-adenosyl-L-homocysteine, a well-known inhibitor of methyltransferase-catalyzed reactions. No GTP gamma S stimulation of methylation was found in cytosolic extracts from any of the other tissues studied, including brain, testis, spleen, and liver, nor in brush-border membranes isolated from the kidney cortex. The methylated proteins were highly sensitive to moderately alkaline conditions, suggesting that the methyl esters were formed on L-isoaspartyl residues and thus methylated by a class II PCMT. These results suggest that class-II-associated protein methylation activity from the soluble fraction of the kidney can be regulated by guanine nucleotides.

Protein carboxyl methylation in kidney brush-border membranes.

Protein carboxyl methylation activity was detected in the cytosol and in purified brush-border membranes (BBM) from the kidney cortex. The protein carboxyl methyltransferase (PCMT) activity associated with the BBM was specific for endogenous membrane-bound protein substrates, while the cytosolic PCMT methylated exogenous substrates (ovalbumin and gelatin) as well as endogenous proteins. The apparent Km for S-adenosyl-L-methionine with endogenous proteins as substrates were 30 microM and 4 microM for the cytosolic and BBM enzymes, respectively. These activities were sensitive to S-adenosyl-L-homocysteine, a well known competitor of methyltransferase-catalyzed reactions, but were not affected by the presence of chymostatin and E-64, two protein methylesterase inhibitors. The activity of both cytosolic and BBM PCMT was maximal at pH 7.5, while BBM-phospholipid methylation was predominant at pH 10.0. Separation of the = methylated proteins by acidic gel electrophoresis in the presence of the cationic detergent benzyldimethyl-n-hexadecylammonium chloride revealed distinct methyl accepting proteins in the cytosol (14, 17, 21, 27, 31, 48, 61 and 168 kDa) and in the BBM (14, 60, 66, 82, and 105 kDa). Most of the labelling was lost following electrophoresis under moderately alkaline conditions, except for a 21 kDa protein in the cytosol and a 23 kDa protein in the BBM fraction. These results suggest the existence of two distinct PCMT in the kidney cortex: a cytosolic enzyme with low selectivity and affinity, methylating endogenous and exogenous protein substrates, and a high-affinity BBM-associated methylating activity.

Tyrosine protein kinase activity in renal brush-border membranes.

Tyrosine protein kinase (TPK) activity was detected in rat renal brush-border membranes (BBM) using poly(Glu80Na,Tyr20) as a substrate. Maximal TPK activity required prior detergent dispersion of the membranes with 0.05% Triton X-100 and the presence of vanadate, a potent inhibitor of phosphotyrosine protein phosphatases, in the phosphorylation medium. Optimal conditions for measurement of TPK activity were 10 mM of MgCl2 and MnCl2, at 30 degrees C and pH 7.0. TPK activity was inhibited by genistein, with a IC50 value of 15 microM, while no inhibition was observed in the presence of 1-(5-isoquinolinesulfonyl)-2-methyl-piperazine dihydrochloride (H7), an inhibitor of serine-threonine kinases. TPK activity was enriched 4-fold in the BBM fraction relative to cortex homogenate. It was co-enriched with BBM enzyme markers, but not with those of the basolateral membrane (BLM). The endogenous substrates of TPK in brush-border and basolateral membranes were determined by Western blot analysis using an antiphosphotyrosine monoclonal antibody (PY20). Various phosphotyrosine-containing proteins were found in the BBM (31, 34, 46, 50, 53, 72, 90, 118 and 170 kDa) and in the BLM (37, 48, 50, 53, 72, 90, 130 and 170 kDa). Addition of exogenous insulin receptor to BBM and BLM increased the phosphorylation of most of the substrates. Solubilization of the TPK activity from BBM with 0.5% CHAPS and subsequent gel filtration on Superdex 75 yielded two peaks of tyrosine protein kinase activity with apparent molecular masses of 49 and 66 kDa. These results provide evidence for a non-receptor TPK activity associated with the renal tubular luminal membrane.

Rapid activation of matrix metalloproteinase-2 by glioma cells occurs through a posttranslational MT1-MMP-dependent mechanism.

Matrix metalloproteinase-2 (MMP-2) has been suggested to play a crucial role in tumor invasion and angiogenesis. In order to understand the mechanisms underlying proMMP-2 activation, we compared the biochemical and cellular events triggered by two potent MMP-2 activators, the lectin concanavalin A (ConA) and the cytoskeleton disrupting agent cytochalasin D (CytoD). Incubation of U87 human glioma cells for 24 h in the presence of ConA or CytoD induced a marked activation of proMMP-2 and this activation was correlated in both cases with an increase in the mRNA levels of MT1-MMP. At the protein level, proMMP-2 activation induced by CytoD or ConA strongly correlated with the appearance of a 43-kDa MT1-MMP proteolytic breakdown product in cell lysates. Interestingly, CytoD also induced a very rapid (2 h) activation of proMMP-2 that was independent of protein synthesis. Under these conditions, CytoD also promoted the rapid proteolytic breakdown of the 63 kDa pro form of MT1-MMP, resulting in the appearance of the 43 kDa MT1-MMP processed form. Overexpression of a recombinant full-length MT1-MMP protein in glioma cells resulted in the activation of proMMP-2 that was correlated with the generation of the 43 kDa fragment of the protein. By contrast, overexpression of the protein in COS-7 cells promoted proMMP-2 activation without inducing the production of the 43 kDa fragment. These results thus suggest that activation of proMMP-2 occurs through both translational and post-translational mechanisms, both involving proteolytic processing of membrane-associated MT1-MMP. This processing of MT1-MMP is, however, not essential to proMMP-2 activation but may represent a regulatory mechanism to control the activity of MT1-MMP.

Matrix metalloproteinase inhibition by green tea catechins.

We have investigated the effects of different biologically active components from natural products, including green tea polyphenols (GTP), resveratrol, genistein and organosulfur compounds from garlic, on matrix metalloproteinase (MMP)-2, MMP-9 and MMP-12 activities. GTP caused the strongest inhibition of the three enzymes, as measured by fluorescence assays using gelatin or elastin as substrates. The inhibition of MMP-2 and MMP-9 caused by GTP was confirmed by gelatin zymography and was observed for MMPs associated with both various rat tissues and human brain tumors (glioblastoma and pituitary tumors). The activities of MMPs were also measured in the presence of various catechins isolated from green tea including (-)-epigallocatechin gallate (EGCG), (-)-epicatechin gallate(ECG), (-)-epigallocatechin (EGC), (-)-epicatechin (EC) and (+)-catechin (C). The most potent inhibitors of these activities, as measured by fluorescence and by gelatin or casein zymography, were EGCG and ECG. GTP and the different catechins had no effect on pancreatic elastase, suggesting that the effects of these molecules on MMP activities are specific. Furthermore, in vitro activation of proMMP-2 secreted from the glioblastomas cell line U-87 by the lectin concanavalin A was completely inhibited by GTP and specifically by EGCG. These results indicate that catechins from green tea inhibit MMP activities and proMMP-2 activation.

Studies on pancreatic acinar cells in tissue culture: basal lamina (basement membrane matrix promotes three-dimensional reorganization.

Rat pancreatic acinar cells have been dissociated and maintained in culture under specific conditions which allow the retention of their differentiated state and three-dimensional organization. When cultured on a basal lamina (basement membrane) matrix, the cells first formed large monolayer patches and then reorganized themselves into acini-like structures. The cells regained their polarity around luminal spaces which appeared to be sealed off by well developed junctional complexes. Typical microvilli appeared at the "apical" plasma membrane projecting themselves into the luminal spaces. The intracellular organization resembled that of the cells in situ: a well developed rough endoplasmic reticulum located towards the "base" of the cell around a nucleus; a supranuclearly positioned Golgi apparatus and numerous secretory granules located in the "apical" region of the cell. Immunocytochemistry has revealed the presence of two pancreatic enzymes, amylase and chymotrypsinogen, in the various cellular compartments involved in secretion; the rough endoplasmic reticulum and Golgi cisternae as well as in the secretory granules. Biochemical evaluations have also shown the presence of amylase in the acinar cells and culture medium. These results thus demonstrate that dissociated pancreatic acinar cells maintained in culture under specific conditions reaggregate themselves into acini-like structures and retain their differentiated morphology as well as their ability to secrete.

P-glycoprotein is localized in caveolae in resistant cells and in brain capillaries.

A significant proportion of P-glycoprotein (P-gp) and caveolin was co-localized in caveolae isolated from resistant (CH(R)C5) cells overexpressing P-gp and from drug-sensitive Chinese hamster ovary cells (AuxB1). The proportion of P-gp and caveolin associated with caveolar microdomains was higher in CH(R)C5 cells grown in the presence of P-gp substrates (cyclosporin A or colchicine) than in untreated CH(R)C5 cells. Coimmunoprecipitation of P-gp and caveolin from CH(R)C5 lysates suggests that there is a physical interaction between them. Furthermore, co-localization of P-gp and caveolin was found in caveolae from brain capillaries, indicating that this association also takes place in vivo.

Activation of the extracellular signal-regulated protein kinase (ERK) cascade by membrane-type-1 matrix metalloproteinase (MT1-MMP).

The mechanisms underlying membrane-type-1 matrix metalloproteinase (MT1-MMP)-dependent induction of cell migration were investigated. Overexpression of MT1-MMP induced a marked increase in cell migration, this increase being dependent on the presence of the cytoplasmic domain of the protein. MT1-MMP-dependent migration was inhibited by a mitogen-activated protein kinase kinase 1 inhibitor, suggesting the involvement of the extracellular signal-regulated protein kinase (ERK) cascade in the induction of migration. Accordingly, MT1-MMP overexpression induced the activation of ERK, this process being also dependent on the presence of its cytoplasmic domain. MT1-MMP-induced activation of both migration and ERK required the catalytic activity of the enzyme as well as attachment of the cells to matrix proteins. The MT1-MMP-dependent activation of ERK was correlated with the activation of transcription through the serum response element, whereas other promoters were unaffected. Taken together, these results indicate that MT1-MMP trigger important changes in cellular signal transduction events, leading to cell migration and to gene transcription, and that these signals possibly originate from the cytoplasmic domain of the protein.

Cellular and subcellular expression of Golf/Gs and Gq/G11 alpha-subunits in rat pancreatic endocrine cells.

We studied the cellular and subcellular localization of Galpha-subunits in pancreas by immunocytochemistry. Golfalpha and G11alpha were specifically localized in islet insulin B-cells and glucagon A-cells, respectively. Gsalpha and Gqalpha labeling was more abundant in B-cells. The presence of Golfalpha in B-cells was confirmed by in situ hybridization. In B-cells, Golfalpha and Gsalpha were found in the Golgi apparatus, plasma membrane (PM) and, remarkably, in mature and immature insulin secretory granules, mainly at the periphery of the insulin grains. Gqalpha was detected on the rough endoplasmic reticulum (RER) near the Golgi apparatus. In A-cells, the Galpha-subunits were mostly within the glucagon granules: G11alpha gave the strongest signal, Gsalpha less strong, Gq was scarce, and Golf was practically absent. Gqalpha and Gsalpha immunoreactivity was detected in acinar cells, although it was much weaker than that in islet cells. The cell-dependent distribution of the Galpha-subunits indicates that the stimulatory pathways for pancreatic function differ in acinar and in islet B- and A-cells. Furthermore, the G-protein subunits in islet cell secretory granules might be functional and participate in granule trafficking and hormone secretion.

Electron spectroscopic imaging for high-resolution immunocytochemistry: use of boronated protein A.

In the present study we adapted electron spectroscopic imaging (ESI) for high-resolution immunocytochemistry. To accomplish this, we applied boronated protein A (B-pA) for indirect detection of specific antigenic sites using pre-embedding and post-embedding protocols. Isolated acinar cells were exposed to wheat germ agglutinin (WGA) and anti-WGA, followed by B-pA, to reveal WGA binding sites at the level of the plasma membrane. The cells were then embedded in Epon and unstained ultra-thin sections were examined by electron microscopy using the ESI mode. For post-embedding, ultra-thin sections of glutaraldehyde-fixed, Lowicryl-embedded pancreatic tissue were exposed to specific antibodies (anti-insulin or anti-amylase), followed by B-pA. The unstained sections were examined using the ESI mode. In both cases, boron was detected with high resolution either at the level of the plasma membrane of acinar cells, demonstrating WGA binding sites, or over secretory granules in pancreatic insulin-secreting cells or acinar cells, demonstrating insulin and amylase, respectively. These findings were compared to those obtained with the protein A-gold technique, and have demonstrated the analogy of both types of labeling. In addition, several control experiments assessed this novel approach. They have demonstrated the specificity of labeling and the high reactivity of B-pA, as well as its antibody-binding properties. Finally, electron energy loss spectral analysis confirmed the presence of boron in the tissue sections at sites where immunolabeling was detected. These results demonstrate that ESI is an appropriate approach for cytochemistry. Since the technique is based on detection of elements, spatial resolution is considered to be in the magnitude of 0.5 nm, which represents a major improvement in resolution over actual electron microscopic cytochemical techniques.

Alterations of vitronectin and its receptor alpha(v) integrin in the rat renal glomerular wall during diabetes.

Vitronectin, a multifunctional glycoprotein present in blood and extracellular matrix, is not only a member of the cell adhesion molecules, but also a regulator of proteolytic enzyme cascades, thereby providing a unique regulatory factor for proteolytic degradation of extracellular matrix and tissue remodeling. Vitronectin interacts with the cell surface through integrins of the alpha(v)-related system. Because vitronectin and its receptor may have a role in various renal physiological and pathological processes, we evaluated their expression in renal tissues of streptozotocin-induced short- and long-term hyperglycemic rats by applying quantitative immunoelectron microscopy and Western blot analysis. Vitronectin was shown over the glomerular basement membrane (GBM) and mesangial matrix (MM), whereas alpha(v) was located along the plasma membrane of endothelial, epithelial, and mesangial cells. Although distribution patterns of vitronectin and alpha(v) integrin labeling in renal tissues from short- and long-term hyperglycemic rats, as well age-matched normoglycemic rats, were similar, increases in their immunoreactive sites were detected in hyperglycemic conditions. Changes also were present in old compared with young normoglycemic animals. The diabetes-related increase in vitronectin was more significant in the GBM than MM, whereas the increase in alpha(v) integrin was as significant in podocytes as mesangial cells. Western blot analysis, performed on isolated glomerular material from normoglycemic and hyperglycemic animals, confirmed those changes. Our results suggest that vitronectin and its receptor, alpha(v) integrin, must have defined roles in molecular mechanisms involved in the pathogenesis of both diabetic and aging nephropathy.

Expression differences in mitochondrial and secretory chaperonin 60 (Cpn60) in pancreatic acinar cells.

In pancreatic acinar cells, chaperonin Cpn60 is present in all the cellular compartments involved in protein secretion as well as in mitochondria. To better understand the role Cpn60 plays in pancreatic secretion, we have evaluated its changes under experimental conditions known to alter pancreatic secretion. Quantitative protein A-gold immunocytochemistry was used to reveal Cpn60 in pancreatic acinar cells. Cpn60 immunolabelings in cellular compartments involved in secretion were found to decrease in acute pancreatitis as well as upon stimulation of secretion and in starvation conditions. A major increase in Cpn60 was recorded in diabetic condition. This was normalized by insulin treatment. Although in certain situations changes in secretory enzymes and in Cpn60 correlate well, in others, nonparallel secretion seemed to take place. In contrast, expression of mitochondrial Cpn60 in acinar cells appeared to remain stable in all conditions except starvation, where its levels decreased. Expression of Cpn60 in the secretory pathway and in mitochondria thus appears to behave differently, and Cpn60 in the secretory pathway must be important for quality control and integrity of secretion.

Differences in secretory granule content in pancreatic acinar cells from peri-insular and tele-insular regions.

By applying morphometrical and quantitative double immunocytochemical techniques, differences in size and in amylase and chymotrypsinogen contents were found among pancreatic zymogen granules. These differences were present in granules of peri-insular and tele-insular acinar cells, the peri-insular ones displaying higher numbers of granules of smaller sizes. No correlation was found among enzyme contents in individual granules, nor was there a correlation between enzyme content and granule size. The results suggest that each individual secretory granule is formed in an independent way and that each enzyme is processed and packaged into granules independently. The differences among granules may be associated with nonparallel secretion, since this phenomenon has been reported in the intracellular processing of secretory enzymes. This hypothesis, however, remains to be demonstrated.

AE-941 (Neovastat): a novel multifunctional antiangiogenic compound.

AE-941 (Neovastat) is a naturally occurring product extracted from cartilage and has antiangiogenic properties. It has reached Phase III clinical trial evaluation for the treatment of solid tumors (non-small cell lung cancer and renal cell carcinoma) and a pivotal Phase II clinical trial in multiple myeloma is ongoing. AE-941 inhibits several steps of the angiogenesis process, including matrix metalloproteinase activities and VEGF signaling pathways. Moreover, AE-941 induces endothelial cell apoptosis and tissue-type plasminogen activator activity, thus suggesting that it is a multifunctional antiangiogenic drug. Results from Phase I/II clinical trials indicate that AE-941, given orally, is well tolerated. Moreover, the median survival time in patients with renal cell carcinoma and non-small cell lung cancer was significantly longer in patients receiving high doses of AE-941 compared to low doses.

Matrix proteinase inhibition by AE-941, a multifunctional antiangiogenic compound.

BACKGROUND: Matrix metalloproteinases (MMPs) play an important role in tissue remodeling under normal physiological and pathological conditions and are thus attractive targets for both diagnostic and therapeutic purposes. Here, we examined the effect of AE-941, an orally bioavailable standardized extract made of cartilage that shows significant antiangiogenic and antimetastatic properties in vivo, on the activity of various members of the MMP family. MATERIALS AND METHODS: The effect of AE-941 on the activity of MMPs was assessed by fluorimetric assays and by substrate gel zymography. RESULTS: AE-941 markedly inhibits the gelatinolytic activity of MMP-2 and to a lesser extent those of MMP-1, MMP-7, MMP-9 and MMP-13. AE-941 also inhibited the elastinolytic activities of MMP-2 and MMP-9 as well as MMP-12 (metalloelastase), porcine pancreatic elastase (PPE), and human leukocyte elastase (HLE). Western blot analysis revealed the presence within AE-941 of immunoreactive TIMP-like proteins, suggesting that these proteins may be at least partly responsible for the observed MMP inhibition. CONCLUSIONS: Taken together, these results demonstrate that AE-941 contains TIMP-like proteins that could be responsible for the specific inhibition of MMPs. Given the recent studies suggesting the presence within this compound of specific inhibitor(s) of endothelial cell proliferation, AE-941 appears as a pleotropic agent able to interfere with several biochemical steps leading to angiogenesis and to other physiopathological conditions. Since AE-941 is currently under Phase III clinical investigations, these findings are also of considerable importance for our understanding of its anticancer properties.

Effects of chrysotile asbestos on DNA synthesis and growth of human embryonic lung fibroblasts.

The in vitro effects of asbestos fibers on thymidine (TdR3H) incorporation and growth of lung fibroblasts have been studied. Incubation of human embryonic lung fibroblasts with UICC Chrysotile B asbestos for 48 hr caused a 3 to 5-fold increase of TdR3H incorporation as compared with control cultures. This increase was dose-dependent with optimal effect obtained with doses as low as 10 micrograms/ml and with cell density of 5 X 10(4) fibroblasts per culture. However, enhanced TdR3H incorporation in treated cells was not correlated with an overall increase of the fibroblast population compared with control cultures as evidenced by cell counts and microscopic examination. Fibroblasts exposed to relatively low concentrations of UICC chrysotile (5-10 micrograms/ml) displayed an initial decrease in cell number compared to controls during the first 24 hr of incubation. At 48 hr however, enhanced TdR3H incorporation occurred with a concomittant increase in cell number. Moreover, continuous exposure of fibroblast cultures to chrysotile (10 micrograms/ml) for a longer period of time led to sustained increase of TdR3H incorporation and resumption of cell proliferation. It is suggested that increased thymidine incorporation is directly related to the effectiveness of asbestos in inhibiting the growth of lung fibroblasts and that measurement of TdR3H incorporation may represent a sensitive means of assessing rapidly the biological activity of asbestos. The possible relevance of this activity to asbestos-induced fibrogenesis and tumorigenesis is also discussed.

Platelet-activating factor stimulates interleukin-6 production by human endothelial cells and synergizes with tumor necrosis factor for enhanced production of granulocyte-macrophage colony stimulating factor.

The interaction between human endothelial cells (EC) and leukocytes during inflammation is in part mediated through the release of soluble factors. Since platelet-activating factor (PAF) is a potent mediator of inflammatory responses, we investigated the potential of PAF to modulate IL-6 and GM-CSF production by EC. Exposure of these cells to PAF resulted in a concentration-dependent increase in IL-6 production, with a maximum at 10(-10) M PAF. Sequential incubation of EC with PAF and TNF alpha resulted in a synergistic increase of IL-6 production. This effect was specific for PAF since it was prevented by preincubation with the PAF receptor antagonist, WEB 2086. Northern blot analysis revealed enhanced IL-6 mRNA expression in PAF-treated EC. However, the synergy observed in protein synthesis between PAF and TNF alpha was not reflected in IL-6 mRNA accumulation, suggesting a post-translational modulation. Pretreatment of EC with the protein synthesis inhibitor cycloheximide before their exposure to PAF resulted, after washout of the cycloheximide, in a markedly augmented production of IL-6, suggesting a synergy between augmented IL-6 mRNA accumulation by PAF and IL-6 mRNA superinduction by cycloheximide. GM-CSF production by EC was also stimulated by the combined effects of PAF and TNF alpha, but PAF alone did not affect GM-CSF production. Taken together, our data suggest that PAF can stimulate EC to synthesize cytokines, including IL-6 and GM-CSF, which may contribute to local and, possibly, systemic responses during inflammation.

Image classification using spectral and spatial information based on MRF models.

A new criterion for classifying multispectral remote sensing images or textured images by using spectral and spatial information is proposed. The images are modeled with a hierarchical Markov Random Field (MRF) model that consists of the observed intensity process and the hidden class label process. The class labels are estimated according to the maximum a posteriori (MAP) criterion, but some reasonable approximations are used to reduce the computational load. A stepwise classification algorithm is derived and is confirmed by simulation and experimental results.