Circulating glycated albumin and glomerular anionic charges.

Aiming to discern the mechanisms by which circulating glycated albumin alters the glomerular filtration properties that lead to glomerular dysfunction in diabetes, the authors studied the distribution and densities of anionic charges through the rat glomerular wall upon intravascular infusion of Amadori products, as well as in various conditions of increased glomerular permselectivity. Polylysine-gold was used as the probe to reveal the anionic charges. The study was carried on renal tissue sections of bovine serum albumin (BSA)- and glycated BSA-injected, normoglycemic animals. Results were generated through morphometrical evaluations of the gold labeling. Changes in glomerular anionic distribution were corroborated on renal tissue sections of short- and long-term diabetic rats and of normal newborn rats, situations known for abnormal glomerular filtration. Altered renal function in these conditions was clearly associated with changes in glomerular anionic charges. On the other hand, the infusion of glycated albumin in the circulation of normal rats, though altering glomerular filtration properties, did not modify the distribution and density of the polylysine-gold labeling through the glomerular basement membrane. Thus, anionic charges seem not to be the factor involved in the early changes of glomerular permeability induced by circulating glycated albumin.

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.

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.

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.

Calmodulin inhibitors trigger the proteolytic processing of membrane type-1 matrix metalloproteinase, but not its shedding in glioblastoma cells.

Most transmembrane proteins are subjected to limited proteolysis by cellular proteases, and stimulation of cleavage of membrane proteins by calmodulin (CaM) inhibitors was recently shown. The present study investigated the ability of several CaM inhibitors to induce the proteolytic cleavage of the membrane type-1 matrix metalloproteinase (MT1-MMP) from the cell surface of highly invasive U-87 glioblastoma cells. Although no shedding of a soluble MT1-MMP form was induced by CaM inhibitors in the conditioned media, we showed that these inhibitors induced MT1-MMP proteolytic processing to the 43 kDa membrane-bound inactive form that was not correlated with an increase in proMMP-2 activation but rather with an increase in tissue inhibitor of MMPs (TIMP)-2 expression levels. Moreover, this proteolytic processing was sensitive to marimastat suggesting the involvement of MMPs. Interestingly, CaM inhibitors antagonized concanavalin A- and cytochalasin D-induced proMMP-2 activation, and affected the cytoskeletal actin organization resulting in the loss of migratory potential of U-87 glioblastoma cells. Cytoplasmic tail-truncated MT1-MMP constructs expressed in COS-7 cells were also affected by CaM inhibitors suggesting that these inhibitors stimulated MT1-MMP proteolytic processing by mechanisms independent of the CaM-substrate interaction. We also propose that TIMP-2 acts as a negative regulator of MT1-MMP-dependent activities promoted by the action of CaM inhibitors in U-87 glioblastoma cells.

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.

Localization of membrane-type 1 matrix metalloproteinase in caveolae membrane domains.

Membrane-type 1 matrix metalloproteinase (MT1-MMP) is a membrane-associated MMP that has been recently reported to have a central role in tumour cell invasion. Here we report that both the native and overexpressed recombinant forms of MT1-MMP are highly enriched in low-density Triton X-100-insoluble membrane domains that contain the caveolar marker protein caveolin 1. Moreover, the MT1-MMP-dependent activation of proMMP-2 induced by concanavalin A and cytochalasin D was correlated with the processing of MT1-MMP to its proteolytically inactive 43 kDa fragment in U-87 glioblastoma and HT-1080 fibrosarcoma tumour cell lines; this processing was also preferentially observed within the caveolar fraction. Interestingly, whereas the expression of caveolin 1 had no effect on the MT1-MMP-dependent activation of proMMP-2, its co-expression with MT1-MMP antagonized the MT1-MMP-increased migratory potential of COS-7 cells. Taken together, our results provide evidence that MT1-MMP is preferentially compartmentalized and proteolytically processed in caveolae of cancer cells. The inhibition of MT1-MMP-dependent cell migration by caveolin 1 also suggests that the localization of MT1-MMP to caveolin-enriched domains might have an important function in the control of its enzymic activity.

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.

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.

Tyrosine phosphorylation of the vascular endothelial-growth-factor receptor-2 (VEGFR-2) is modulated by Rho proteins.

The effects of Rho-specific modifying toxins on the tyrosine phosphorylation of endothelial cell proteins were investigated. Incubation of the cells with the Rho-activating toxin cytotoxic necrotizing factor 1 (CNF1) induced a marked increase in the tyrosine phosphorylation of a number of signalling intermediates of the vascular endothelial growth factor (VEGF)-mediated cascade, including focal adhesion kinase, paxillin, phospholipase Cgamma1 and a Shc-associated protein of 195 kDa. Both CNF1- and VEGF-dependent tyrosine phosphorylation of these proteins were significantly reduced by prior incubation with C3 transferase, a known inhibitor of RhoA function, suggesting a Rho-dependent mechanism. The stimulation of endothelial cells with CNF1 resulted in a marked increase in the tyrosine phosphorylation of the VEGF receptor (VEGFR)-2, which was correlated with a stimulation of its kinase activity and with its association with downstream tyrosine phosphorylated proteins. The stimulatory effect of CNF1 was specific for VEGFR-2 since the phosphotyrosine content of VEGFR-1 was not affected by the toxin. Transient overexpression of a dominant-active RhoA mutant also induced an increase in the tyrosine phosphorylation of the VEGFR-2, whereas overexpression of a dominant-inactive form of the protein was without effect. Taken together, these results indicate that Rho proteins may play an important role in angiogenesis by modulating the tyrosine phosphorylation levels of VEGFR-2.

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.

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.

Shark cartilage extracts as antiangiogenic agents: smart drinks or bitter pills?

The use of crude cartilage for the treatment of human cancers remains a subject of controversy. In this brief commentary, we reviewed the current knowledge on the anticancer properties of cartilage. We then presented the properties of AE-941, a novel standardized water-soluble extract derived from shark cartilage that represents less than 5% of the crude cartilage. It is a multifunctional antiangiogenic product that contains several biologically active molecules. EA-941 is one of the few antiangiogenic drugs that is under phase III clinical investigation. It is currently evaluated in Europe and North America for the treatment of refractory renal cell carcinoma and in North America for metastatic non small cell lung cancer.

Regulation of Rho protein binding to membranes by rhoGDI: inhibition of releasing activity by physiological ionic conditions.

The Rho GDP dissociation inhibitor (GDI) is an ubiquitously expressed regulatory protein involved in the cycling of Rho proteins between membrane-bound and soluble forms. Here, we characterized the Rho solubilization activity of a glutathione S-transferase (GST) - GDI fusion protein in a cell-free system derived from rat kidney. Addition of GST-GDI to kidney brush border membranes resulted in the specific release of Cdc42 and RhoA from the membranes, while RhoB and Ras were not extracted. The release of Cdc42 and RhoA by GST-GDI was dose dependent and saturable with about 50% of both RhoA and Cdc42 extracted. The unextracted Rho proteins were tightly bound to membranes and could not be solubilized by repeated GST-GDI treatment. These results demonstrated that kidney brush border membranes contained two populations of RhoA and Cdc42. Furthermore, the GST-GDI solubilizing activity on membrane-bound Cdc42 and RhoA was abolished at physiological conditions of salt and temperature in all tissues examined. When using bead-immobilized GST-GDI, KCl did not reduced the binding of Rho proteins. However, washing brush border membranes with KCl prior treatment by GST-GDI inhibited the extraction of Rho proteins. Taken together, these results suggest that the binding of GDI to membrane-bound Cdc42 and RhoA occurs easily under physiological ionic strength conditions, but a complementary factor is required to extract these proteins from membranes. These observations suggest that the shuttling activity of GDI upon Rho proteins could be normally downregulated under physiological conditions.

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.

Localization of RhoA GTPase to endothelial caveolae-enriched membrane domains.

Caveolae are small microdomains of the plasma membrane that are thought to play important roles in signal transduction processes. In this work, we have investigated the association of Rho proteins with caveolae-enriched membrane domains isolated from cultured endothelial cells. Fractionation of ECV304 cells by sucrose gradient density centrifugation in the absence of detergent resulted in the co-sedimentation of a significant proportion of RhoA and Cdc42 with known caveolae marker proteins, including caveolin, but not with other non-caveolae membrane proteins such as the angiotensin-converting enzyme. Immunoprecipitation experiments carried on crude endothelial cell lysates as well as with solubilized caveolae-enriched membrane domains showed the coimmunoprecipitation of caveolin with RhoA but not with Cdc42. Incubation of endothelial cell lysates with a glutathione-S-transferase (GST)-RhoA fusion protein resulted in the specific precipitation of caveolin, while addition of GST-caveolin-1 to the lysates promoted the precipitation of RhoA. Moreover, incubation of bacterially expressed RhoA with GST-caveolin-1 resulted in the precipitation of RhoA, indicating that RhoA directly interacts with caveolin-1. This interaction was found to be nucleotide-independent and was not affected by prior modification of RhoA with the C3 exoenzyme from C. botulinium or with the cytotoxic necrotinizing factor from E. coli. Taken together, these results suggest the association of RhoA with endothelial caveolae-enriched membrane domains, likely through physical interaction with caveolin-1. These findings may provide new insights into the functions played by Rho proteins and caveolae in signal transduction events.

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.

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.

Purification, cloning, and sequence analysis of a Mr = 30,000 protein from sea urchin axonemes that is important for sperm motility. Relationship of the protein to a dynein light chain.

We have generated a series of monoclonal antibodies against axonemal proteins from sea urchin spermatozoa in order to identify novel proteins involved in the regulation of flagellar motility. The monoclonal antibody D405-14 inhibited the motility of demembranated-reactivated sperm models at low concentrations and recognized a single polypeptide of 33 kDa (p33) on immunoblots of sea urchin axonemal proteins. Fractionation of the axonemes with high salt solutions, heat, and detergent resulted in the selective extraction of p33 into a 0.6 M NaCl-soluble and a 0.5% sodium lauryl sarcosinate (Sarkosyl)-soluble form. Both forms of p33 were purified to apparent homogeneity by immunoaffinity chromatography on monoclonal antibody D405-14-Sepharose. We have also isolated and sequenced a full-length cDNA clone encoding the 33-kDa protein. The sequence predicts a polypeptide of 260 amino acids having a mass of 29,730 Da and an isoelectric point of 9.3. Sequence comparison indicates that p33 is 66% identical (74% similar) to the p28 light chain of axonemal inner dynein arm of Chlamydomonas reinhardtii. Taken together, these results suggest that we have identified a p28 light chain homolog in sea urchin sperm axoneme and that this protein may play a dynamic role in flagellar motility.

Adhesion-independent synergy of monocytes and endothelial cells in cytokine production: regulation of IL-6 and GM-CSF production by PAF.

Co-Cultures of monocytes (MO) and endothelial cells (EC) were studied for their capacity to synergize in the production of interleukin-6 (IL-6) and granulocyte-macrophage colony-stimulating factor (GM-CSF), two cytokines potentially important in vascular physiopathology. Resting monocytes produced detectable amounts of IL-6 but no GM-CSF, whereas confluent EC produced significant quantities of GM-CSF, but minimal IL-6. In co-cultures without stimuli, additive synthesis of both cytokines was observed. When EC were pretreated, however, with either PAF, TNF or both stimuli, before addition of MO, synergistic production of IL-6 was observed. In contrast, GM-CSF production was not enhanced by coculture of monocytes with activated EC. When either cell population was fixed with paraformaldehyde or killed by freeze-thawing before addition to the co-culture, cytokine levels reverted to those produced by the unaffected population alone. On the other hand, separating the two cell populations by a cell-impermeable membrane in transwell cultures did not affect the synergistic production of the cytokines. Taken together, our data suggest that EC and MO can synergize in response to stimuli by producing IL-6 and that this synergy is dependent on the integrity of both cell populations, but independent of cell-cell contact.