Because the role of elemental sulfur in human nutrition has not been studied extensively, it is the purpose of this article to emphasize the importance of this element in humans and discuss the therapeutic applications of sulfur compounds in medicine. Sulfur is the sixth most abundant macromineral in breast milk and the third most abundant mineral based on percentage of total body weight. The sulfur-containing amino acids (SAAs) are methionine, cysteine, cystine, homocysteine, homocystine, and taurine. Dietary SAA analysis and protein supplementation may be indicated for vegan athletes, children, or patients with HIV, because of an increased risk for SAA deficiency in these groups. Methylsulfonylmethane (MSM), a volatile component in the sulfur cycle, is another source of sulfur found in the human diet. Increases in serum sulfate may explain some of the therapeutic effects of MSM, DMSO, and glucosamine sulfate. Organic sulfur, as SAAs, can be used to increase synthesis of S-adenosylmethionine (SAMe), glutathione (GSH), taurine, and N-acetylcysteine (NAC). MSM may be effective for the treatment of allergy, pain syndromes, athletic injuries, and bladder disorders. Other sulfur compounds such as SAMe, dimethylsulfoxide (DMSO), taurine, glucosamine or chondroitin sulfate, and reduced glutathione may also have clinical applications in the treatment of a number of conditions such as depression, fibromyalgia, arthritis, interstitial cystitis, athletic injuries, congestive heart failure, diabetes, cancer, and AIDS. Dosages, mechanisms of action, and rationales for use are discussed. The low toxicological profiles of these sulfur compounds, combined with promising therapeutic effects, warrant continued human clinical trails.

The inflammatory response in the injured spinal cord, an immune privileged site, has been mainly associated with the poor prognosis. However, recent data demonstrated that, in fact, some leukocytes, namely monocytes, are pivotal for repair due to their alternative anti-inflammatory phenotype. Given the pro-inflammatory milieu within the traumatized spinal cord, known to skew monocytes towards a classical phenotype, a pertinent question is how parenchymal-invading monocytes acquire resolving properties essential for healing, under such unfavorable conditions. In light of the spatial association between resolving (interleukin (IL)-10 producing) monocytes and the glial scar matrix chondroitin sulfate proteoglycan (CSPG), in this study we examined the mutual relationship between these two components. By inhibiting the de novo production of CSPG following spinal cord injury, we demonstrated that this extracellular matrix, mainly known for its ability to inhibit axonal growth, serves as a critical template skewing the entering monocytes towards the resolving phenotype. In vitro cell culture studies demonstrated that this matrix alone is sufficient to induce such monocyte polarization. Reciprocal conditional ablation of the monocyte-derived macrophages concentrated at the lesion margins, using diphtheria toxin, revealed that these cells have scar matrix-resolving properties. Replenishment of monocytic cell populations to the ablated mice demonstrated that this extracellular remodeling ability of the infiltrating monocytes requires their expression of the matrix-degrading enzyme, matrix metalloproteinase 13 (MMP-13), a property that was found here to be crucial for functional recovery. Altogether, this study demonstrates that the glial scar-matrix, a known obstacle to regeneration, is a critical component skewing the encountering monocytes towards a resolving phenotype. In an apparent feedback loop, monocytes were found to regulate scar resolution. This cross-regulation between the glial scar and monocytes primes the resolution of this interim phase of spinal cord repair, thereby providing a fundamental platform for the dynamic healing response.

Hyperphosphorylated microtubule-associated protein tau is the major proteinaceous component of the paired helical and straight filaments which constitute a defining neuropathological characteristic of Alzheimer's disease and a number of other neurodegenerative disorders. We have recently shown that full-length recombinant tau assembles into Alzheimer-like filaments upon incubation with heparin. Heparin also promotes phosphorylation of tau by a number of protein kinases, prevents tau from binding to taxol-stabilized microtubules, and produces rapid disassembly of microtubules assembled from tau and tubulin. Here, we have used the above parameters to study the interactions between tau protein and a number of naturally occurring and synthetic glycosaminoglycans. We show that the magnitude of the glycosaminoglycan effects is proportional to their degree of sulfation. Thus, the strongly sulfated glycosaminoglycans dextran sulfate, pentosan polysulfate, and heparin were the most potent, whereas the non-sulfated dextran and hyaluronic acid were without effect. The moderately sulfated glycosaminoglycans heparan sulfate, chondroitin sulfate, and dermatan sulfate had intermediate effects, whereas keratan sulfate had little or no effect. These in vitro interactions between tau protein and sulfated glycosaminoglycans reproduced the known characteristics of paired helical filament-tau from Alzheimer's disease brain. Sulfated glycosaminoglycans are present in nerve cells in Alzheimer's disease brain in the early stages of neurofibrillary degeneration, suggesting that their interactions with tau may constitute a central event in the development of the neuronal pathology of Alzheimer's disease.

The many biocharacteristics of glycosaminoglycans (GAGs) make them valuable molecules to be incorporated in collagenous biomaterials. To prepare tailor-made collagen-GAG matrices with a well-defined biodegradability and (bioavailable) GAG content, the crosslinking conditions have to be controlled. Additionally, the ultrastructural location of GAGs in engineered substrates should resemble that of the application site. Using chondroitin sulfate (CS) as a model GAG, these aspects were evaluated. The methodology was then applied for other GAGs. CS was covalently attached to collagen using 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS). A maximum of about 155 mg CS/g matrix could be immobilized. CS incorporation and bioavailability, as evaluated by interaction with specific antibodies and glycosidases, was dependent on the molar ratio EDC:carboxylic groups of CS. The denaturation temperature could be modulated from 61 to 85 degrees C. The general applicability of EDC/NHS for immobilizing GAGs was demonstrated with dermatan sulfate, heparin, and heparan sulfate. These matrices revealed comparable physico-chemical characteristics, biodegradabilities, and preserved bioavailable GAG moieties. At the ultrastructural level, GAGs appeared as discrete, electron-dense filaments, each filament representing a single GAG molecule. Distribution was independent of GAG type. They were observed throughout the matrix fibers and at the outer sites, and located, either parallel or orthogonally, at the periphery of individual collagen fibrils. Compositional and ultrastructural similarity between matrices and tissue structures like cartilage and basement membranes can be realized after attachment of specific GAG types. It is concluded that EDC/NHS is generally applicable for attachment of GAGs to collagen. Modulation of crosslinking conditions provides matrices with well-defined GAG contents, and biodegradabilities. Ultrastructural similarities between artificially engineered scaffolds and their possible application site may favor the use of specific collagen-GAG matrices in tissue engineering.

The versican (PG-M) gene encodes a chondroitin sulfate proteoglycan that is nonpermissive for cell migration and appears in association with slow cell proliferation and cytodifferentiation. Using the techniques of in situ hybridization and immunocytochemistry on sectioned mouse embryos, we found that the mRNA and protein for versican show similar distributions and are expressed in a dynamic pattern during development of the heart. Versican exhibits generalized expression in the tubular heart but becomes rapidly downregulated in the atrium and exhibits higher transcript levels on the right side of the ventricular chamber than the left, before the onset of ventricular septation. Versican is expressed strongly in the trabeculated ventricular myocardium, whereas the compact proliferative zone has lower transcript abundance. It is expressed in the outer layers and on the crest of the ventricular septum and is prominent on the mesenchymal cap of the primary atrial septum. Versican is particularly strongly expressed in the endocardial cushions of the atrioventricular and outflow tract regions and in the atrioventricular, semilunar, and venous valves. This study raises the possibility that versican may be involved in specification of the ventricular chambers, in growth and fusion of the atrial and ventricular septa, and in the transformation from epithelium to mesenchyme that characterizes development of the endocardial cushions. Versican may be a key participant in cardiogenesis, responding to the many diffusible signals that mediate interactions between the developing endocardium and myocardium.

The hyaluronic acid binding region was prepared by clostripain digestion of chondroitin sulfate proteoglycan isolated from the Swarm rat chondrosarcoma, and biotinylated in the presence of associated hyaluronic acid and link protein. After removal of hyaluronic acid by gel filtration in 4 M guanidine HCl, the biotinylated binding region-link protein complex was used as a specific histochemical probe in conjunction with avidin-peroxidase. Its utility was initially evaluated by comparison with Alcian blue staining of the axial region of 2 to 5 day chick embryos, where staining was seen in the dorsolateral area between the neural tube and the ectoderm, in the perichordal mesenchyme, and in developing limb buds. Light and electron microscopic studies of early postnatal rat cerebellum indicate that hyaluronic acid is primarily localized in the extracellular space of immature brain. Staining specificity was demonstrated by the ability of hyaluronic acid oligosaccharides of appropriate size to block the staining reaction, and by the absence of staining after treatment of tissue sections with protease-free Streptomyces hyaluronidase, which degrades only this glycosaminoglycan.

Light-chain amyloidosis (AL) is characterized by immunoglobulin light-chain fragments aggregating into amyloid fibrils that deposit extracellularly in vital organs such as the kidney, the heart, and the liver, resulting in tissue degeneration and organ failure, leading to death. Cardiac involvement is found in 50% of AL patients and presents the most severe cases with a life expectancy of less than a year after diagnosis. In this study, we have characterized the variable domain of a cardiac AL patient light chain called AL-09. AL-09 folds as a beta-sheet and is capable of forming amyloid fibrils both in the presence of sodium sulfate and in self-seeded reactions under physiological conditions. Glycosaminoglycans such as dermatan sulfate and heparin promote amyloid formation of self-seeded AL-09 reactions, while the glycosaminoglycan chondroitin sulfate A stabilized oligomeric intermediates and did not elongate the preformed fibrils (nucleus) present in the reaction. Finally, the histological dye Congo red, known to bind to the cross beta-sheet structure of amyloid fibrils, inhibits AL-09 amyloid fibril formation in the presence of sodium sulfate and in self-seeded reactions. This paper provides insight into the impact of different reagents on light-chain stability, structure, amyloid fibril formation, and inhibition.

Perineuronal nets (PNNs) are unique extracellular matrix structures that wrap around certain neurons in the CNS during development and control plasticity in the adult CNS. They appear to contribute to a wide range of diseases/disorders of the brain, are involved in recovery from spinal cord injury, and are altered during aging, learning and memory, and after exposure to drugs of abuse. Here the focus is on how a major component of PNNs, chondroitin sulfate proteoglycans, control plasticity, and on the role of PNNs in memory in normal aging, in a tauopathy model of Alzheimer's disease, and in drug addiction. Also discussed is how altered extracellular matrix/PNN formation during development may produce synaptic pathology associated with schizophrenia, bipolar disorder, major depression, and autism spectrum disorders. Understanding the molecular underpinnings of how PNNs are altered in normal physiology and disease will offer insights into new treatment approaches for these diseases.

Mucopolysaccharidosis type VI (MPS VI; Maroteaux-Lamy syndrome) is a lysosomal storage disorder caused by mutations in the N-acetylgalactosamine-4-sulfatase (arylsulfatase B, ARSB) gene. ARSB is a lysosomal enzyme involved in the degradation of the glycosaminoglycans (GAG) dermatan and chondroitin sulfate. ARSB mutations reduce enzyme function and GAG degradation, causing lysosomal storage and urinary excretion of these partially degraded substrates. Disease onset and rate of progression is variable, producing a spectrum of clinical presentation. In this study, 105 MPS VI patients-representing about 10% of the world MPS VI population-were studied for molecular genetic and biochemical parameters. Direct sequencing of patient genomic DNA was used to identify ARSB mutations. In total, 83 different disease-causing mutations were found, 62 of which were previously unknown. The novel sequence changes included: 38 missense mutations, five nonsense mutations, 11 deletions, one insertion, seven splice-site mutations, and four polymorphisms. ARSB mutant protein and residual activity were determined on fibroblast extracts for each patient. The identification of many novel mutations unique to individuals/their families highlighted the genetic heterogeneity of the disorder and provided an appropriate cohort to study the MPS VI phenotypic spectrum. This mutation analysis has identified a clear correlation between genotype and urinary GAG that can be used to predict clinical outcome.

Axon outgrowth between the spinal cord and the hindlimb of the chick embryo is constrained by three tissues that border axon pathways. Growth cones turn to avoid the posterior sclerotome, perinotochordal mesenchyme, and pelvic girdle precursor during normal development and after experimental manipulation. We wanted to know if these functionally similar barriers to axon advance also share a common molecular composition. Since the posterior sclerotome differentially binds peanut agglutinin (PNA) and since PNA binding is also typical of prechondrogenic differentiation, we examined the pattern of expression of PNA binding sites and cartilage proteoglycan epitopes in relation to axon outgrowth. We found that all three barrier tissues preferentially express both PNA binding sites and chondroitin-6-sulfate (C-6-S) immunoreactivity at the time when growth cones avoid these tissues. Moreover, both epitopes are expressed in the roof plate of the spinal cord and in the early limb bud, two additional putative barriers to axon advance. In contrast, neither epitope is detected in peripheral axon pathways. In the somites, this dichotomous pattern of expression clearly preceded the invasion of the anterior sclerotome by either motor growth cones or neural crest cells. However, in the limb, barrier markers disappeared from presumptive axon pathways in concert with the invasion of axons. Since this coordinate pattern suggested that the absence of barrier markers in these axon pathways requires an interaction with growth cones, we analyzed the pattern of barrier marker expression following unilateral neural tube deletions. We found that PNA-negative axon pathways developed normally even in the virtual absence of axon outgrowth. We conclude that the absence of staining with carbohydrate-specific barrier markers is an independent characteristic of the cells that comprise axon pathways. These results identify two molecular markers that characterize known functional barriers to axon advance and suggest that barrier tissues may impose patterns on peripheral nerve outgrowth by virtue of their distinct molecular composition.

Malaria during pregnancy is a major health problem for African women. The disease is caused by Plasmodium falciparum malaria parasites, which accumulate in the placenta by adhering to chondroitin sulfate A (CSA). The interaction between infected erythrocytes and the placental receptor is mediated by a parasite expressed protein named VAR2CSA. A vaccine protecting pregnant women against placental malaria should induce antibodies inhibiting the interaction between VAR2CSA and CSA. Much effort has been put into defining the part of the 350 kDa VAR2CSA protein that is responsible for binding. It has been shown that full-length recombinant VAR2CSA binds specifically to CSA with high affinity, however to date no sub-fragment of VAR2CSA has been shown to interact with CSA with similar affinity or specificity. In this study, we used a biosensor technology to examine the binding properties of a panel of truncated VAR2CSA proteins. The experiments indicate that the core of the CSA-binding site is situated in three domains, DBL2X-CIDR(PAM) and a flanking domain, located in the N-terminal part of VAR2CSA. Furthermore, recombinant VAR2CSA subfragments containing this region elicit antibodies with high parasite adhesion blocking activity in animal immunization experiments.

The objective of this study was to determine how changes in the network structure and properties of hyaluronic acid (HA) hydrogels, due to variations in the macromer molecular weight (50-1,100 kDa) and macromer concentration (2-20 wt %), affect neocartilage formation by encapsulated auricular chondrocytes. To investigate tissue formation, swine auricular chondrocytes were photoencapsulated in the various networks, implanted subcutaneously in the dorsum of nude mice, and explanted after 6 and 12 weeks for biochemical and histological analysis. After 12 weeks, the various constructs were 81-93% water, contained between 0.1 x 10(6) and 0.6 x 10(6) chondrocytes per sample, and consisted of 0-0.049 microg chondroitin sulfate/mug wet weight (glycosaminoglycan (GAG) content) and 0.002-0.060 microg collagen/microg wet weight. Histological staining showed an even distribution of chondrocytes and GAGs in addition to minimal type I collagen staining and intense and uniform type II collagen staining in the constructs with greatest neocartilage production. Hydrogels fabricated from 2 wt % of the 50 kDa HA macromer most resembled the properties of native cartilage and show the greatest promise for continued development for cartilage regeneration.

The main types of noncollagenous ECM proteins in liver are FN, laminin, PGs and elastin. Also present is a pure carbohydrate polymer, hyaluronic acid. Their concentrations increase during fibrogenesis in a specific sequence and spatial distribution. FN is among the first to appear. The accumulation of matrix proteins in the space of Disse (perisinusoidal fibrosis) is most important for some clinical consequences of fibrosis. Perisinusoidal FSC are the cell type responsible for exaggerated ECM formation at sites of tissue injury, since these cells synthesize at a high rate in vitro all the noncollagenous matrix components occurring in fibrotic liver matrix (FN, laminin, dermatan sulfate, chondroitin sulfate, hyaluronic acid) and since FSC proliferate and transform into myofibroblast-like cells with an enhanced matrix protein synthesis rate. Recent experimental data suggest strong cellular cooperation of these cells with activated liver macrophages or Kupffer cells, platelets, and regenerating hepatocytes, which leads via a paracrine mechanism involving TGF beta, TGF alpha/EGF, PDGF, and probably other cytokines to stimulated proteoglycan synthesis, proliferation, and transformation into myofibroblast-like cells. The latter cell type has the potential of auto-stimulation (by an autocrine process) involving secretion of TGF beta and TGF alpha/EGF. Based on these findings, a model of FSC activation is proposed (Fig. 10).

OBJECTIVE

This work studied the presence of inflammatory and atherogenic lipoprotein markers that could explain the high incidence of cardiovascular disease (CVD) reported in rheumatoid arthritis (RA) patients.

METHODS

Inflammatory markers were 1) soluble adhesion molecules (intercellular adhesion molecule [ICAM] and vascular cell adhesion molecule [VCAM]), 2) C-reactive protein (CRP), 3) fibrinogen (Fb), 4) cytokines (interferon-gamma [IFNgamma], tumor necrosis factor alpha [TNFalpha]), and 5) secretory group IIA phospholipase A2 (sPLA2-IIA). Atherogenic lipoprotein markers were 1) the size distribution of plasma lipoprotein subclasses, and 2) the binding affinity of low-density lipoprotein (LDL) to chondroitin 6-sulfate glycosaminoglycan (GAG).

RESULTS

RA patients (n = 31) and matched controls (n = 28) had similar plasma concentrations of total cholesterol, triglycerides, Apo B, Apo A-I, very low-density lipoprotein, intermediate-density lipoprotein, and high-density lipoprotein (HDL). RA patients had significantly higher plasma levels of sPLA2-IIA, ICAM, CRP, Fb, TNFalpha, and IFNgamma compared with controls. RA patients also had significantly higher levels of small, dense LDL-1 (P < 0.05) and lower levels of small HDL-2 particles (P < 0.001) compared with controls. In addition, LDL from RA patients had a significantly higher binding affinity (Kd) to GAG (mean +/- SD Kd 204+/-22.4 nM Apo B) than did LDL from control subjects (Kd 312+/-36 nM Apo B) (P < 0.05). This Kd value showed a significant negative correlation with the plasma levels of LDL-1 (r = -0.566, P < or = 0.004). In RA patients, a significant positive correlation was obtained between sPLA2-IIA and CRP, ICAM, and LDL-1. HDL-2 showed a negative correlation with sPLA2-IIA.

CONCLUSIONS

These atherogenic lipoprotein factors combined with the presence of chronic inflammation may contribute to the high CVD-related mortality in RA patients.

This study focuses on the effects of simulated microgravity (0g) on the human follicular thyroid carcinoma cell line ML-1. Cultured on a three-dimensional clinostat, ML-1 cells formed three-dimensional MCTSs (MCTS diameter: 0.3 +/- 0.01 mm). After 24 and 48 h of clinorotation, the cells significantly decreased fT3 and fT4 secretion but up-regulated the thyroid-stimulating hormone-receptor expression as well as the production of vimentin, vinculin, and extracellular matrix proteins (collagen I and III, laminin, fibronectin, chondroitin sulfate) compared with controls. Furthermore, ML-1 cells grown on the clinostat showed elevated amounts of the apoptosis-associated Fas protein, of p53, and of bax but showed reduced quantities of bcl-2. In addition, signs of apoptosis became detectable, as assessed by terminal deoxynucleotidyl transferase-mediated dUTP digoxigenin nick end labeling, 4', 6-diamidino-2-phenylindole staining, DNA laddering, and 85-kDa apoptosis-related cleavage fragments. These fragments resulted from enhanced 116-kDa poly(ADP-ribose)polymerase (PARP) activity and apoptosis. These observations suggest that clinorotation elevates intermediate filaments, cell adhesion molecules, and extracellular matrix proteins and simultaneously induces apoptosis in follicular thyroid cancer cells. In conclusion, our experiments could provide a regulatory basis for the finding that astronauts show low thyroid hormone levels after space flight, which may be explained by the increase of apoptosis in thyrocytes as a result of simulated 0g.

We investigated the function of cyclin-dependent kinase 2 (Cdk2) in neural progenitor cells during postnatal development. Chondroitin sulfate proteoglycan (NG2)-expressing progenitor cells of the subventricular zone (SVZ) show no significant difference in density and proliferation between Cdk2(-/-) and wild-type mice at perinatal ages and are reduced only in adult Cdk2(-/-) mice. Adult Cdk2(-/-) SVZ cells in culture display decreased self-renewal capacity and enhanced differentiation. Compensatory mechanisms in perinatal Cdk2(-/-) SVZ cells, which persist until postnatal day 15, involve increased Cdk4 expression that results in retinoblastoma protein inactivation. A subsequent decline in Cdk4 activity to wild-type levels in postnatal day 28 Cdk2(-/-) cells coincides with lower NG2+ proliferation and self-renewal capacity similar to adult levels. Cdk4 silencing in perinatal Cdk2(-/-) SVZ cells abolishes Cdk4 up-regulation and reduces cell proliferation and self- renewal to adult levels. Conversely, Cdk4 overexpression in adult SVZ cells restores proliferative capacity to wild-type levels. Thus, although Cdk2 is functionally redundant in perinatal SVZ, it is important for adult progenitor cell proliferation and self-renewal through age-dependent regulation of Cdk4.

BACKGROUND

The objectives of this study were to (1) examine patient treatment preferences for knee osteoarthritis, (2) determine the influence of specific medication characteristics on patients' choices, and (3) examine whether patient preferences are consistent with current practice.

METHODS

A total of 100 consecutive patients with symptomatic knee osteoarthritis completed an interactive computer questionnaire administered during a face-to-face interview. We measured the relative impact of specific medication characteristics (including administration, risks, benefits, and cost) on patients' choice, and the percentage of patients preferring nonselective nonsteroidal antiinflammatory drugs (NSAIDs), cyclooxygenase-2 inhibitors, glucosamine and/or chondroitin sulfate, opioid derivatives, and capsaicin across varying risks, benefits, and costs.

RESULTS

Of the characteristics studied, variation in the risk of common adverse effects and gastrointestinal ulcer had the greatest impact on patients' choice. Assuming patients are responsible for the full cost of their medications, over 40% prefer capsaicin. Cyclooxygenase-2 inhibitors become patients' preferred choice only if they are described as being 3 times as effective as capsaicin and are covered by insurance. Nonselective NSAIDs are among the least preferred options across all simulations.

CONCLUSIONS

When evaluating multiple alternatives, many older patients with knee osteoarthritis are willing to forgo treatment effectiveness for a lower risk of adverse effects. The patient treatment preferences derived in this study conflict with the current widespread use of nonselective NSAIDs in older patients with arthritis.

The connective tissue stroma of malignant tumors is a newly formed tissue that supports the growth and progression of neoplastic cells. Proteoglycans are intrinsic components of this complex structure and molecular changes in this class of macromolecules can significantly affect behavioral properties of transformed cells. We report that human colon carcinoma contained increased levels of a chondroitin sulfate proteoglycan that exhibited an altered glycosaminoglycan structure in which 0- and 6-sulfated units, as detected by specific monoclonal antibodies, predominated. Proteoglycans with such epitopes were localized primarily to the connective tissue stroma surrounding the tumor cells but not to the tumor cells themselves or the native, non-cancerous connective tissue. Analysis of mRNA encoding PG-40, the main chondroitin sulfate proteoglycan of colon tissue, revealed a 7-fold increase in the two transcripts encoding this gene product. This increase was evident whether the data were normalized to total RNA content or beta-actin mRNA levels. The altered steady state levels of PG-40 mRNA did not correlate with any significant gene amplification or rearrangement of PG-40 in human colon cancer. However, when genomic DNA was tested for degree of methylation, the colon carcinoma tissue showed a marked hypomethylation of PG-40 gene locus, a finding that has been associated with increased gene activation. Interestingly, PG-40 gene was also hypomethylated in cultured colon fibroblasts, which express PG-40, but not in colon carcinoma cells which do not express this gene. These results indicate that specific proteoglycan changes occur in colon carcinoma and that these alterations are the product of stromal cells that are topologically associated with and functionally respondent to the growing malignant cells. This is the first evidence that enhanced PG-40 expression in a human malignant tissue is associated with a hypomethylated gene and suggests that the control of PG-40 gene expression may represent an important factor in the progression of colon carcinoma.

Versican is a large extracellular chondroitin sulfate proteoglycan that belongs to the family of lecticans. Alternative splicing of versican generates at least four isoforms named V0, V1, V2, and V3. We show here that ectopic expression of versican V1 isoform induced mesenchymal-epithelial transition (MET) in NIH3T3 fibroblasts, and inhibition of endogenous versican expression abolished the MET in metanephric mesenchyme. MET in NIH3T3 cells was demonstrated by morphological changes and dramatic alterations in both membrane and cytoskeleton architecture. Molecular analysis showed that V1 promoted a "switch" in cadherin expression from N- to E-cadherin, resulting in epithelial specific adhesion junctions. V1 expression reduced vimentin levels and induced expression of occludin, an epithelial-specific marker, resulting in polarization of V1-transfected cells. Furthermore, an MSP (methylation-specific PCR) assay showed that N-cadherin expression was suppressed through methylation of its DNA promoter. Exogenous expression of N-cadherin in V1-transfected cells reversed V1's effect on cell aggregation. Reduction of E-cadherin expression by Snail transfection and siRNA targeting E-cadherin abolished V1-induced morphological alteration. Transfection of an siRNA construct targeting versican also reversed the changed morphology induced by V1 expression. Silencing of endogenous versican prevented MET of metanephric mesenchyme. Taken together, our results demonstrate the involvement of versican in MET: expression of versican is sufficient to induce MET in NIH3T3 fibroblasts and reduction of versican expression decreased MET in metanephric mesenchyme.

The Costello syndrome is characterized by prenatally increased growth, postnatal growth retardation, coarse face, loose skin resembling cutis laxa, nonprogressive cardiomyopathy, developmental delay, and a outgoing, friendly behavior. Patients can develop papillomata, especially around the mouth, and have a predisposition for malignancies (mainly abdominal and pelvic rhabdomyosarcoma in childhood). Costello syndrome is likely to be an autosomal dominant disorder. The pathogenesis is unclear, but there are many clues for a disturbed elastogenesis, possibly through a disturbed elastin-binding protein reuse by chondroitin sulfate-bearing proteoglycans accumulation. A review of the findings in the 73 patients that have been described in sufficient detail is provided.

The receptors responsible for avid alveolar macrophage (AM) phagocytosis of unopsonized environmental particulates have not been well defined. This study used flow cytometry to quantitate the effects of a panel of soluble ligands for macrophage adhesion receptors on AM binding of unopsonized environmental dusts (titanium dioxide, TiO2; iron oxide, Fe2O3; alpha-quartz, SiO2; diesel engine exhaust dust) or fluorescent latex beads. Polyanionic ligands of the macrophage scavenger receptor (SR) for acetylated-LDL caused marked inhibition of AM binding of the oxide particles and latex beads (e.g., TiO2 binding; polyinosinic acid (polyl), 10 micrograms/ml: 70.2 +/- 1.5% inhibition, mean +/- SE, n = 11). In contrast, no inhibition was seen with the polyanions heparin and chondroitin sulfate (chond-S), or dextran, consistent with the known inhibitor profile of macrophage SRs for acetylated-LDL AM uptake of latex or SiO2 beads instilled into lungs of hamsters was inhibited by administration of polyl but not chondroitin sulfate (AM beads per cell: control, 6.1 +/- 0.7; polyl, 3.5 +/- 0.2; chond-S, 5.1 +/- 0.7, n>> or = 4, p < 0.05 for control vs polyl) indicating macrophages SRs operate in vivo as well as in vitro. In contrast, AM binding of the carbonaceous diesel dust particles was not inhibited by any ligand tested. AM uptake of unopsonized TiO2, SR ligands or acetylated LDL caused no significant activation of AM respiratory burst or TNF production, consistent with past observations that opsonin-independent phagocytosis of inert particles by normal AMs is not accompanied by pro-inflammatory activation. These data implicate macrophage-type SRs in AM binding of charged environmental particles and indicate that distinct mechanisms mediate binding of carbonaceous dusts.

In this paper, we identify the chondroitin sulfate proteoglycan versican V2 as a major inhibitor of axonal growth in the extracellular matrix of the mature central nervous system. In immunohistochemical and in situ hybridization experiments we show that this tissue-specific splice variant of versican is predominantly present in myelinated fiber tracts of the brain and in the optic nerve, most likely being expressed by oligodendrocytes. We demonstrate that isolated versican V2 strongly inhibits neurite outgrowth of central and peripheral neurons in stripe-choice assays using laminin-1 as permissive substrate. The inhibitory character of versican V2 is maintained after removal of chondroitin sulfate and N- and O-linked oligosaccharide side chains, but it is abolished after core protein digestion with proteinase-K. Our data support the notion, that intact versican V2 prevents excessive axonal growth during late phases of development and hereby participates in the structural stabilization of the mature central nervous system.

A rat chondrosarcoma cell line and primary bovine chondrocytes have been used to study cell-mediated aggrecan catabolism. Addition of 1 microM retinoic acid to chondrosarcoma cultures resulted in aggrecan proteolysis with the release of greater than 90% of the cell layer aggrecan into the medium within 4 days. NH2-terminal sequencing of chondroitin sulfate-substituted catabolic products gave a single major NH2-terminal sequence of ARGNVILTXK, initiating at Ala374. This showed that the proteinase, commonly referred to as "aggrecanase," which cleaves the Glu373-Ala374 bond of the interglobular domain of aggrecan (Sandy, J. D., Neame, P. J., Boynton, R. E., and Flannery, C. R. (1990) J. Biol. Chem. 266, 8683-8685), is active in this cell system. Aggrecan G1 domain, generated by cleavage of the interglobular domain, was also liberated during catabolism and this was characterized with three antipeptide antisera. Anti-CDAGWL was used as a general probe for G1 domain. Anti-FVDIPEN was used to specifically detect G1 domain with COOH terminus of Asn341, the form which is readily generated by cleavage of aggrecan by a wide range of matrix metalloproteinases. Anti-NITEGE antiserum was used to specifically detect G1 domain with COOH terminus of Gln373, the form which is the expected product of "aggrecanase"-mediated cleavage of aggrecan. Western blot analysis indicated that a single form of G1 domain of about 60 kDa was formed. G1 domain of this size reacted with both anti-CDAGWL and anti-NITEGE but not with anti-FVDIPEN. Similar experiments with primary bovine chondrocyte cultures, treated with either retinoic acid or interleukin 1, showed that two forms of catabolic G1 domain, of about 62 and 66 kDa, were formed. Both of these forms reacted on Western blots with anti-CDAGWL and also with anti-NITEGE. It is suggested that cell-mediated catabolism of the aggrecan interglobular domain in these culture systems, whether promoted by retinoic acid or interleukin 1, primarily involves cleavage of the Glu373-Ala374 bond by aggrecanase. The accumulation of G1 domain with a COOH-terminal of Glu373 shows that such aggrecanase-mediated cleavage can occur independent of the cleavage of the Asn341-Phe342 bond by matrix metalloproteinases.