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The purpose of this study was (1) to confirm an experimental model of aminophylline-induced necrotizing enterocolitis (NEC); and (2) to determine whether nuclear magnetic resonance (NMR) imaging, based upon proton relaxation values (T1, T2), could detect NEC during its early pathogenesis. Sixty male weanling Lewis rats (avg wt = 75 g) were randomly assigned to one of three experimental groups: (A) superior mesenteric artery (SMA) occlusion (1 min) + aminophylline treatment (40 mg/kg); (B) SMA occlusion; and (C) sham midline laparotomy (control). All surviving animals were sacrificed at 48 hr postoperation and a specimen of ileum was removed for light microscopy (LM), electron microscopy (EM), and NMR analysis. Percentage water content was determined for representative specimens. Mortality occurred only in experimental group A animals (18.2%; P less than 0.05), who had received aminophylline. Microscopy of ileum from sacrificed animals of this group showed changes ranging from mild cellular disruption to severe hemorrhagic necrosis. Early ultrastructural changes consistent with NEC were detectable with EM before LM. Proton relaxation results obtained with NMR showed significant prolongation of T1 (252.5 +/- 4.4 msecs; P less than 0.001) and T2 (69.3 +/- 1.4 msecs; P less than 0.025) during the first stages of NEC. NMR may indeed enable early, safe diagnostic imaging of NEC in infants receiving aminophylline, or those who are otherwise at increased risk for development of this disease.

Two cases of large, multiple splenic hamartomas in children with pancytopenia, bone marrow hyperplasia, lymphadenopathy, hepatosplenomegaly, frequent infections, growth retardation, and fever are reported. These symptoms were relieved by splenectomy, and have not recurred during follow-up periods of one year and nine years. The sharply circumscribed lesions comprised large portions of the resected spleens and were composed of dilated vascular channels filled with mononuclear cells and immunoblasts. The lesions lacked splenic cords or trabeculae, lymphoid follicles, Reed-Sternberg cells, and granulomas or other evidence of infection. Splenic hamartomas are usually single small lesions found incidentally at necropsy or laparotomy. Splenic hamartomas associated with symptoms and hypersplenism are large, and often confluent multiple tumors. Recognition of their benign nature is important in light of the current practice of laparotomy for staging and diagnosis of malignant conditions.

Cell surface proteoglycans are strategically positioned to regulate interactions between cells and their surrounding environment. Such interactions play key roles in several biological processes, such as cell recognition, adhesion, migration, and growth. These biological functions are in turn necessary for the maintenance of differentiated phenotype and for normal and neoplastic development. There is ample evidence that a special type of proteoglycan bearing heparan sulfate side chains is localized at the cell surface in a variety of epithelial and mesenchymal cells. This molecule exhibits selective patterns of reactivity with various constituents of the extracellular matrix and plasma membrane, and can act as growth modulator or as a receptor. Certainly, during cell division, membrane constituents undergo profound rearrangement, and proteoglycans may be intimately involved in such processes. The present work will focus on recent advances in our understanding of these complex macromolecules and will attempt to elucidate the biosynthesis, the structural diversity, the modes of cell surface association, and the turnover of heparan sulfate proteoglycans in various cell systems. It will then review the multiple proposed roles of this molecule, with particular emphasis on the binding properties and the interactions with various intracellular and extracellular elements. Finally, it will focus on the alterations associated with the neoplastic phenotype and will discuss the possible consequences that heparan sulfate may have on the growth of normal and transformed cells.

To characterize the electromechanical effects of acute and 2-week-old cryoinjury, programmed stimuiation and epicardial M-mode echo mapping (7.5 mHz) were performed prior to, at 15 minutes and 35 ± 2 days after cryoinjury in 10 dogs. Epicardial and intramural bipolar and unipoiar electrograms were recorded in five of the dogs. Cryoinjury was produced with a 5 mm in diameter flat cryoprobe at -60°C applied to the left ventricular epicardium for 10 minutes at each of six contiguous sites. Cryoinjury, acutely and at 2 weeks, was characterized by a loss of normal intramural systolic thickening and a decrease in echo density. The maximum depth of cryoinjury determined by echo ranged from .55 to .85 cm acutely, and .50 to .80 cm chronically and it correlated acutely and chronically (r = .80; r = .85) with pathologically documented depth of cryoinjury at 2 weeks. Acutely, the presence of either an abnormal intramural unipolar or bipolar electrogram also defined the depth of cryoinjury within .16 cm. In contrast, epicardiai electrograms were not useful for judging depth of cryoinjury. A QS complex on the epicardial unipolar electrogram was recorded over the cryoinjury acutely and at 2 weeks in four of five dogs despite preserved subendocardial systolic wall thickening and normal histology extending 40 to 65% of the wall thickness. Epicardiai bipolar eiectrograms uniformly showed a marked decrease in amplitude and siope of the intrinsic deflection, a small hut insignificant increase in width and no multicomponent activity. Ventricular fibrillation was reproducibly initiated with three ventricular extrastimuli in five of 10 dogs at 2 weeks. No ventricular tachycardia was initiated. Thus, cryoinjury produces characteristic echogram and electrogram changes that are immediate and persistent. The distinct echo signature as well as the presence of abnormal intramural electrograms can be used to judge the depth of cryoinjury. Cryoinjury does not potentiate the inducibility of ventricular tachycardia. The significance of the inducibility of ventricular fibrillation cannot be determined. Our cryoinjury model can be used to characterize electrograms which represent recordings of myocardial depolarization through an interface of cellular necrosis and early fibrosis.

Pure sarcomas of the esophagus are exceedingly rare. We report a case of esophageal synovial sarcoma which occurred in an adolescent. The tumor was locally resected, sparing the patient esophagectomy. After postoperative radiation therapy, the patient remains alive and well without evidence of disease 28 mo after operation. The unique nature of polypoid sarcoma of the esophagus, and the potential for cure without esophagectomy, is discussed.

Previous studies have shown that human colon carcinomas contain elevated amounts of chondroitin sulfate proteoglycan (CS-PG) and hyaluronic acid, and that the major site of synthesis of these products is the host mesenchyme surrounding the tumor. These findings have led to the proposal that the abnormal formation of the tumor stroma is modulated by the neoplastic cells. The experiments of this paper were designed to explore further this complex phenomenon in an in vitro system using co-cultures of phenotypically stable human colon smooth muscle (SMC) and carcinoma cells (WiDr). The results showed a 3-5-fold stimulation of CS-PG and hyaluronic acid biosynthesis in the co-cultures as compared to the values predicted from the individual cell type cultured separately. The increase in CS-PG was not due to changes in specific activity of the precursor pool, but was rather due to a net increase in synthesis, inasmuch as it was associated with neither a stimulation of cell proliferation nor with an inhibition of intracellular breakdown. These biochemical changes were corroborated by ultrastructural studies which showed a marked deposition of proteoglycan granules in the co-cultures. Several lines of evidence indicated that the SMC were responsible for the overproduction of CS-PG: i) SMC synthesized primarily CS-PG when cultured alone, in contrast to the WiDr, which synthesized exclusively heparan sulfate proteoglycan; ii) only the SMC in co-culture stained with an antibody raised against the amino terminal peptide of a CS-PG (PG-40), structurally and immunologically related to that synthesized by the SMC; iii) the stimulation of CS-PG in SMC could be reproduced, though to a lesser extent, using medium conditioned by WiDr, whereas medium conditioned by SMC had no effects on WiDr. In conclusion this study has reproduced in vitro a tumor-associated matrix with a proteoglycan composition similar to that observed in vivo and provides further support to the concept that production of a proteoglycan-rich extracellular environment is regulated by specific tumor-host cell interactions.

The effects of ethanol on the loss of the human sperm acrosome, as determined by the chlortetracycline fluorescence assay and by indirect immunofluorescence assay, were assessed over 6 hours during incubation at 37 C in BWW medium containing 0 to 250 mM ethanol. Both assays gave the same results. At the end of 6 hours, 48 +/- 6% acrosomal loss was found in samples in 250 mM ethanol compared with 4 +/- 1% in the absence of ethanol. After 0.25 hour, the first time point chosen for sampling, the spermatozoa in 250 mM ethanol showed 23 +/- 3% loss of acrosomes compared with less than 1% in the absence of ethanol. Ultrastructural studies revealed that the ethanol-treated spermatozoa showed complete acrosomal loss as well as loss of the equatorial segment. No examples of the vesiculation characteristic of the physiologic acrosome reaction were found in the 150 cells examined. Calcium is required for the ethanol-mediated acrosomal loss: omission of Ca2+, addition of 2 mM EGTA, or 0.2 mM verapamil blocked the effect. Ethanol induced a dose-dependent efflux of cholesterol from human spermatozoa, but the ethanol-induced acceleration of acrosomal loss occurred to the same extent in the presence of cholesterol microdispersions that prevented this efflux. The loss of the equatorial segment, which is necessary for egg penetration, during ethanol-induced acrosomal loss would explain the known effect of ethanol in inhibiting, rather than enhancing, the penetration of zona-free hamster eggs by human spermatozoa.

Summary The major proteoglycan in bovine parietal pericardium is a low molecular weight dermatansulfate proteoglycan. It possesses structural and immunologic characteristics similar to those of the small proteoglycan found in tendon. We demonstrate that digestion of purified pericardial proteoglycan with low levels of Vg protease results in the liberation of the glycosaminoglycan chain and of a 40 kDa resistant fragment. A similar 40 kDa fragment can be obtained by Vg protease digestion of the proteoglycan deglycosylated by chondroitinase ABC. Although the protein core size of the pericardial proteoglycan is similar to that of tendon PG II, the size of the glycosaminoglycan chain liberated from the former is smaller. The pericardial proteoglycan and its V8 protease products reacted with an anti-PG II antiserum by immunoblotting. The anti-PG-II antibody localized in the pericardial tissue by the immunoperoxidase technique. The presence of intrachain disulfide bonds in the structure of pericardial proteoglycan core protein and V8 resistant fragment was demonstrated by their decreased electrophoretic mobility after disulfide reduction. Digestion of pericardial proteoglycan with Cathepsin C resulted in a rapid liberation of the glycosaminoglycan chain from the core protein, indicating that its attachment site was very close to the amino terminus. Ultrastructural examination of pericardial tissue utilizing Cuprolinic Blue revealed a periodic association of the proteoglycan with the dle band on the collagen fibrils. Electron microscopic immunohistochemical studies confirmed the perifibrillar association of pericardial proteoglycan. The present data demonstrate that, although the pericardial proteoglycan possesses some unique structural features, it shares structural and immunological characteristics to place it in the category of the small PG II family.

The biosynthesis of proteoglycans in short term organ culture of human colon and colon carcinoma was studied. Proteoglycans, labeled with [35S]sulfate and [3H]serine, were extracted with either 4 M or 0.5 M guanidine HCl in the presence of protease inhibitors and sequentially purified by associative and dissociative CsCl density gradient ultracentrifugation. Normal colon synthesized two polydisperse classes of proteoglycans: a large heparan sulfate-containing monomer, with a Kav of 0.48 on Sepharose CL-2B and a small dermatan sulfate-containing monomer with a Kav of 0.65. A portion (25%) of the proteoglycans was found as aggregate when chromatographed under associative conditions, and the larger monomers interacted with hyaluronic acid to an extent greater than the smaller proteoglycans. Following papain or alkali treatment, the free glycosaminoglycan side chains of both monomers eluted as a single broad peak (Kav = 0.5) from Sepharose CL-6B, with an estimated Mr of 20 X 10(3). In contrast, colon carcinoma synthesized only one proteoglycan monomer, which aggregated to a limited extent (12%). This proteoglycan population, with a Kav of 0.7 on CL-2B, contained chondroitin sulfate as the major glycosaminoglycan (greater than 81%), with small amounts of dermatan sulfate. The glycosaminoglycans had an estimated Mr of 9 X 10(3), and the disaccharides released by chondroitinase ABC consisted of 32% 4-sulfate and 68% 6-sulfate. Electron microscopy of mixed proteoglycancytochrome c monolayers from the associative fractions of normal and neoplastic colon revealed aggregated complexes which were similar in over structure, although smaller than the proteoglycan aggregates from cartilage.

The postmortem findings in a patient with the MURCS association (müllerian duct aplasia/hypoplasia, renal agenesis or ectopy, and cervicothoracic somite dysplasia) are reported. This is the first autopsy study since the syndrome was recognized. The autopsy revealed abnormalities of the venous, pulmonary, and central nervous systems that had not been reported previously in patients with this syndrome. A review of the literature suggested that although the MURCS association usually occurs sporadically, as in this case, a familial association is occasionally present. In some cases the MURCS association may be a genetically determined pleiotropic condition.

We have investigated the biosynthesis of heparan sulfate proteoglycan in human colon carcinoma cells cultured in either the absence of L-glutamine or the presence of 6-diazo-5-oxo-L-norleucine (DON), a glutamine analogue. Following a 24-h incubation with 100 micrograms/ml DON (0.58 mM) or without L-glutamine, the incorporation of [35S]sulfate was maximally inhibited to about 50%, whereas the incorporation of [3H]leucine or [3H]serine and their specific activity were not significantly affected. Several lines of evidence indicate that the inhibition of [35S]sulfate incorporation was mediated by a reduction in the intracellular pool of UDP-N-acetylhexosamine: the intracellular hexosamine levels were reduced by about 50%; taking into account the changes in specific activity, the incorporation of [3H]glucosamine was also significantly inhibited; and exogenous D-glucosamine (2.3 mM) was capable of substantially reversing the inhibitory effects of DON. This decrease in endogenous sugar supply resulted in the generation of an underglycosylated, lower buoyant density proteoglycan which contained significantly fewer heparan sulfate chains of otherwise normal size and sulfation and even fewer O-linked oligosaccharides. These biochemical changes were corroborated ultrastructurally by the appearance of smaller ruthenium red-stained proteoglycans on the surface of the cells. DON also caused a marked inhibition of cell proliferation and profound morphological changes, both of which were reversible upon culturing in DON-free medium. These results demonstrate that perturbations in glutamine metabolism have profound effects on the structure of heparan sulfate proteoglycan and on the phenotype of human colon carcinoma cells and indicate that DON treatment could be useful for studying post-translational modifications of proteoglycans in various cell systems.

The immunologic localization of catalase in human eosinophilic leukocytes was studied by use of rabbit anti-human erythrocyte catalase (aHEC) antiserum. Indirect immunofluorescence studies revealed intense localization of fluorescence in the cytoplasm of the eosinophils with a pattern of distribution corresponding to that of the eosinophil granules. Control samples and other nucleated blood elements remained essentially unreactive. Immunoelectron microscopy using aHEC coupled to protein A-gold complexes revealed that catalase was present exclusively within the cytoplasmic granules and equally distributed over the paracrystalline body and the homogeneous granule matrix. The presence of catalase within the paracrystalline body has not been demonstrated before with conventional cytochemical techniques. The results raise the possibility that catalase is stored in the paracrystalline body in an inactive or latent form readily available during eosinophil activation and granule release.

In addition to containing Type IV collagen, laminin and entactin, basement membranes contain small amounts of proteoglycans substituted primarily with heparan sulfate chains. We have previously shown, however, that parietal yolk sacs in organ culture synthesize predominantly chondroitin sulfate proteoglycan. In the present study, we have used histochemical and immunohistochemical techniques coupled with chondroitinase ABC digestion to provide evidence for the presence of chondroitin sulfate proteoglycan in the basement membrane (Reichert's membrane) of the 14.5-day rat embryo parietal yolk sac. The results revealed numerous cuprolinic blue-positive filaments and granules, 20–30 nm in greater length or diameter, dispersed throughout the thickness of the basement membrane. Both structures were removed by preincubating freshly isolated parietal yolk sacs with chondroitinase ABC. A similar labeling pattern was also obtained with immunoelectron microscopy using gold-labeled monoclonal anti-bodies directed against the three major isomers of protein-bound chondroitin sulfate. In contrast, coarser cuprolinic blue granules, 40–100 nm in diameter, were neither sensitive to chondroitinase ABC digestion nor labeled by the monoclonal antibodies. These results thus indicate that Reichert's membrane contains chondroitin sulfate proteoglycan in addition to heparan sulfate proteoglycan.

Human colon carcinoma (HT-29) cells were examined for their capacity to bind and respond to 1,25-dihydroxycholecalciferol [1,25-(OH)2D3]. These cells are known to differentiate and increase their population doubling time when galactose is substituted for glucose in their media. High-affinity and specific binding of 1,25-(OH)2[3H]D3 was observed in extracts of these cells grown in glucose. The binder sedimented in sucrose gradients and eluted from DEAE-cellulose columns in a manner indistinguishable from rabbit intestinal 1,25-(OH)2D3-receptor. Smaller amounts of this binder were seen in HT-29 cells grown in galactose. Both glucose-fed and galactose-fed cells exhibited a dose-dependent decrease in growth rate on exposure to 10(-12) to 10(-6) mol/L 1,25-(OH)2D3. Ultrastructural examination of galactose-fed and glucose + 1,25-(OH)2D3-treated cells showed enterocytic differentiation and features that were not distinguishable between these groups. Sucrase activity was higher in galactose-fed cells and did not change with 1,25-(OH)2D3 treatment. However, the lower sucrase activity in glucose-fed cells increased after exposure to 10(-8) mol/L 1,25-(OH)2D3. These results indicate receptor content and bioresponsivity to 1,25-(OH)2D3 in a human enterocytic cell line, suggesting that it will be a useful model for the study of the mechanisms of action of this sterol.

We provide direct evidence for the presence of unsulfated, but fully elongated heparan glycosaminoglycans covalently linked to the protein core of a heparan sulfate proteoglycan synthesized by human colon carcinoma cells. Chemical and enzymatic studies revealed that a significant proportion of these chains contained glucuronic acid and N-acetylated glucosamine moieties, consistent with N-acetylheparosan, an established precursor of heparin and heparan sulfate. The presence of unsulfated chains was not dependent upon the exogenous supply of sulfate since their synthesis, structure, or relative amount did not vary with low exogenous sulfate concentrations. Culture in sulfate-free medium also failed to generate undersulfated heparan sulfate-proteoglycan, but revealed an endogenous source of sulfate which was primarily derived from the catabolism of the sulfur-containing amino acids methionine and cysteine. Furthermore, the presence of unsulfated chains was not due to a defect in the sulfation process because pulse-chase experiments showed that they could be converted into the fully sulfated chains. However, their formation was inhibited by limiting the endogenous supply of hexosamine. The results also indicated the coexistence of the unsulfated and sulfated chains on the same protein core and further suggested that the sulfation of heparan sulfate may occur as an all or nothing phenomenon. Taken together, the results support the current biosynthetic model developed for the heparin proteoglycan in which unsulfated glycosaminoglycans are first elongated on the protein core, and subsequently modified and sulfated. These data provide the first evidence for the presence of such an unsulfated precursor in an intact cellular system.

The cellular and molecular mechanisms regulating the reversible accumulation of nonhelical, underhydroxylated procollagen in the rough endoplasmic reticulum (RER) remain obscure. To clarify these mechanisms, we isolated chondrocytes from chick vertebral cartilage and kept them in scorbutic monolayer cultures. By Day 9 of culture, the chondrocytes had accumulated a large amount of underhydroxylated Type II procollagen in their RER. Within 1 h of ascorbate treatment, the accumulated procollagen was hydroxylated; this was accompanied by a slight stimulation of procollagen secretion and was followed by a marked stimulation starting between 2 and 3 h of treatment. Secretion of the accumulated procollagen was completed by about 24 h of treatment. Strikingly, the marked stimulation of procollagen secretion at 2-3 h of treatment was associated with marked remodeling of the RER. This organelle came to consist of a few, unusually large cisternae ("sacs") and many flat cisternae while the RER in untreated cells consisted of uniform, oval cisternae. The RER remodeling was accompanied by a comparable redistribution of the accumulated Type II procollagen stored in it. The RER sacs and flat cisternae invariably communicated directly and were still detectable by 8 h but not by 24 h of treatment. RER remodeling and procollagen redistribution also occurred in untreated chondrocytes that had been shifted to 23 degrees C for 2-3 h. Together, the data indicate that folding of the accumulated procollagen molecules into their normal helical configuration is followed by procollagen redistribution within, and remodeling of, the RER. These processes may have a role in stimulating procollagen export from the RER and secretion.

In 28 Dorsett sheep, ligation of the distal homonymous (equivalent to human left anterior descending) and second diagonal coronary arteries produced a constant transmural infarct of 22.9% +/- 2.5% (mean +/- standard deviation) of the left ventricular mass. Serial left ventriculograms showed that within four hours the infarct segment expands, wall thickness decreases, and aneurysmal dilatation occurs and progresses over the next 60 days in all sheep. Epicardial ventricular point references indicated that adjacent noninfarcted myocardium participates in the formation of the aneurysm. Anatomy of the coronary vasculature was studied in 22 excised sheep hearts. In sheep, coronary arterial anatomy is remarkably constant. The left coronary artery provides all of the blood supply to the left ventricle and septum and only a small rim of both the anterior and posterior right ventricles. Cardiac veins from the left ventricle drain into the coronary sinus, which also receives the left azygos vein. Right ventricular veins drain separately. The essentially separate coronary circulations to the two ventricles, the paucity of coronary collateral circulation, and the consistent evolution of left ventricular infarcts into aneurysms are important advantages of the ovine model for both metabolic and ventricular mechanical studies of acute myocardial infarction and left ventricular aneurysm.

The metabolism of heparan sulfate proteoglycan, a major product of human colon carcinoma cells, was investigated in a series of pulse-chase experiments using a combination of quantitative biochemistry and electron microscope autoradiography. This was possible primarily because these cells incorporate [35S]sulfate exclusively into heparan sulfate proteoglycan, thus allowing the possibility of correlating the two sets of information. The results showed a progressive movement of the newly synthesized proteoglycan from the Golgi to the cell surface, where it became closely associated with the plasma membrane and was labeled ultrastructurally by both ruthenium red and radiosulfate. Subsequently, about 55% was released into the medium (t1/2 approximately 2.5 h) where it resided as intact macromolecule and was neither endocytosed nor degraded further. The remaining 45% was internalized and converted into smaller species through a series of degradative steps. Initially (Step 1) there was proteolytic cleavage of the protein core and partial endoglycosidic cleavage of the heparan sulfate chains (t1/2 approximately 6 h), with generation of larger glycosaminoglycan-peptide intermediates with chains of Mr approximately 10,000, about one-third their original size. These components were subsequently converted (Step 2) to yet smaller, limiting fragments of Mr approximately 5,000, which were finally depolymerized (Step 3) with quantitative release of free sulfate. The intracellular degradation of the proteoglycan, particularly Steps 2 and 3, was markedly inhibited by choloroquine, implicating the involvement of acidic compartments in the catabolism of these macromolecules. This was corroborated by the autoradiographic studies which showed the close association of 35S-labeled products with secondary lysosomes. However, the initial degradation of the proteoglycan might have occurred in a prelysosomal compartment since Step 1 was not totally blocked by chloroquine. The combined results indicate that the intracellular degradation of heparan sulfate follows structural as well as functional compartmentalization and provide a model that may be shared by other cell systems.

The embryonic rat parietal yolk sac has been previously shown to synthesize a number of basement membrane glycoconjugates including type IV procollagen, laminin, and entactin. In this study, parietal yolk sacs were isolated from 14.5-day rat embryos and incubated in organ culture for 4-7 h with [35S]sulfate, [3H] glucosamine, and/or 3H-labeled amino acids, and the newly synthesized proteoglycans were characterized. The major [35S]sulfate-labeled macromolecule represented approximately 90% of the medium and 80% of the tissue radioactivity. It also represented nearly 80% of the total [3H]glucosamine-labeled glycosaminoglycans. After purification by sequential ion-exchange chromatography and isopycnic CsCI density gradient ultracentrifugation, size-exclusion high-performance liquid chromatography showed a single species with an estimated Mr of 8-9 X 10(5). The intact proteoglycan did not form aggregates in the presence of exogenous hyaluronic acid or cartilage aggregates. Alkaline borohydride treatment released glycosaminoglycan chains with Mr of 2.0 X 10(4) which were susceptible to chondroitinase AC II and chondroitinase ABC digestion. Analysis by high-performance liquid chromatography of the disaccharides generated by chondroitinase ABC digestion revealed that chondroitin 6-sulfate was the predominant isomer. The uronic acid content of the glycosaminoglycans was 92% glucuronic acid and 8% iduronic acid, and the hexosamine content was 96% galactosamine and 4% glucosamine. No significant amounts of N- or O-linked oligosaccharides were detected. Deglycosylation of the proteoglycan with chondroitinase ABC in the presence of protease inhibitors revealed a protein core with an estimated Mr of 1.25-1.35 X 10(5). These results indicated that the major proteoglycan synthesized by the 14.5-day rat embryo parietal yolk sac is a high-density chondroitin sulfate containing small amounts of copolymeric dermatan sulfate. Hyaluronic acid and minor amounts of heparan sulfate proteoglycan were also detected.

The subcellular localization of human skin chymase to mast cell granules was established by immunoelectron microscopy, and binding of chymase to the area of the dermo-epidermal junction, a basement membrane, was demonstrated immunocytochemically in cryosections incubated with purified proteinase prior to immunolabeling. Because heparin and heparan sulfate proteoglycans are major constituents of mast cell granules and basement membranes, respectively, the ability of chymase to bind to glycosaminoglycans (GAG) was investigated. Among a variety of GAGs, only binding of chymase to heparin and heparan sulfate appears physiologically significant. Binding was ionic strength-dependent, involved amino groups on the proteinase, and correlated with increasing GAG sulfate content, indicating a predominantly electrostatic association. Interaction with heparin was observed in solutions containing up to 0.5 M NaCl, and interaction with heparan sulfate was observed in solutions containing up to 0.3 M NaCl. Binding of heparin did not detectably affect catalysis of peptide substrates, but may reduce accessibility of proteinase to protein substrates. Measurements among a series of serine class proteinases indicated that heparin binding was a more common property of mast cell proteinases than proteinases stored in other secretory granules. Binding of chymase to heparin is likely to have a storage as well as a structural role within the mast cell granule, whereas binding of chymase to heparan sulfate may have physiological significance after degranulation.

We demonstrate that the cell surface heparan sulfate proteoglycan of human colon carcinoma cells has an affinity for a hydrophobic matrix. This property is mediated by sequences in the core protein, since papain-or alkaline borohydride-released heparan sulfate chains do not bind to the matrix. Trypsin releases a [ 3 H]leucine-rich, unsulfated, hydrophobic peptide, with M r ~ 5000. This domain is present in neither the proteoglycan released into the medium nor in the intracellular degradation products. It is proposed that this peptide may represent the portion of the core protein intercalated into the plasma membrane.

Biosynthetic and proliferative characteristics of tubulointerstitial fibroblasts probed with paracrine cytokines. Fibroblasts in parenchymal organs potentially contribute extracellular matrix to local fibrogenic processes. This contribution, in some circumstances, may be initiated by cytokines disseminated from inflammatory lesions. Different populations of fibroblasts, however, might respond distinctively to this cytokine bath depending on the microenvironment in which they reside. We have begun to explore this issue using syngeneic, low-passage fibroblasts cultured in serum-free media that were derived originally from the dermis (DFBs) and from tubulointerstitium (TFBs) of the kidney. Our findings indicate that, while fibroblasts from each compartment appear similar at the ultrastructural level, there are a variety of functional differences which distinguish their proliferative response, and their collagen secretory response (types I, III, IV, and V) following challenge with various doses of immune-relevant cytokines (TGFβ, EGF, IL-1, IL-2 and γIFN) in culture. DFBs, for example, express more surface EGF receptors than do TFBs, and, as a consequence, exhibit a more robust proliferative response to EGF in serum-free media. Unstimulated DFBs also secrete more collagen types I and III than TFBs, while unstimulated TFBs secrete more types IV and V. The expression of these collagens in TFBs was confirmed by Northern blot hybridization. When these sets of fibroblasts were further stimulated by cytokines, some of the cytokines not only differentially effect the secretion of various species of collagens within the same group of cells, but also between cells from populations which are anatomically distinct. DFBs, furthermore, at mid-level doses of cytokine, demonstrated a general trend towards less secretion of all types of collagen (particularly for TGFβ, EGF, and IL-2), while TFBs seemed less repressive. In TFBs the cytokine-induced responses for collagen types I and III tended to be discordant, and for types I and IV EGF inhibited, while TGFβ stimulated the secretory process. These findings speak collectively for the presence of a functional heterogeneity among organ-based populations of syngeneic fibroblasts in normal tissues.

There is a growing realization that the whole tumor cell-matrix complex must be investigated in order to fully understand the process of cancer growth and metastasis. Proteoglycans are intrinsic constituents of the cell surface, extracellular matrix, and basement membrane, three logistically and functionally important structures involved in most cellular interactions. Proteoglycans influence the behavior of normal and malignant cells by virtue of their expanded configuration, polyanionic nature and, most of all, by their ability to interact with a variety of cellular products. Consequently, they have been implicated in a number of biological processes including proliferation, recognition, adhesion, and migration. They can serve as links between the extracellular and intracellular environment and thus transduce key biological signals. They can act as receptors for interstitial collagens and other matrix proteins and thus contribute to the organization of pericellular matrix. During neoplastic development there is a profound structural rearrangement of these macromolecules at both the plasma membrane and the pericellular level. Qualitative and quantitative abnormalities in proteoglycan metabolism may contribute to the establishment of some well-known neoplastic properties, including lack of cohesiveness, abnormal assembly of extracellular matrix, abnormal growth, and invasion. The present work will focus on recent advances in our understanding of these complex macromolecules and on some of the alterations associated with the neoplastic phenotype, and will then attempt to elucidate some of the mechanisms regulating these changes.

The cellular localization of apolipoprotein E (apo-E) in normal and estrogen-treated perfused rabbit livers was studied using monospecific antibodies against purified apo-E. Indirect immunofluorescence revealed intense linear deposits along the hepatic plasma membrane of normal animals and this pattern was unchanged when the livers were perfused with very low density (VLDL), low density lipoproteins (LDL), or buffer alone. Surprisingly, estrogen-treated animals revealed granular apo-E immunoreactive sites within the hepatocytes, suggesting rapid internalization of these proteins. These results raise the possibility that estrogen selectively stimulates the internalization of apo-E.

The intercellular matrix is a highly organized, dynamic system that dictates the overall shape and structure of organs. The spatial and chemical signals which are present in the extracellular matrix modulate the phenotypic expression of epithelial and mesenchymal cells. In turn, these cells regulate the chemistry and structural organization of the extracellular matrix. This active interplay between cells and their products leads to the formation of a highly complex environment in which normal and neoplastic cells can thrive and proliferate. Proteoglycans are suitable candidates for mediating this constant exchange of information. For instance, they influence the diffusion of molecules across tissues, the migration of cells along defined pathways, the surface properties of cells, and the structure of biological filters. These effects are primarily mediated by the polyanionic nature of the proteoglycan, by their expanded configuration in tissues and body fluids, and by their ability to interact with a variety of important matrix macromolecules. They are indeed constituents of both the stromal matrix and the cell surface (Fig. 1), and their biosynthesis, secretion and metabolism are highly regulated by intrinsic and extrinsic signals. These molecules are composed of a protein backbone to which a number of glycosaminoglycan chains and oligosaccharides are covalently attached, much like the branches to a tree. The proteoglycan-rich intercellular matrix can thus be conceived as a microcosmic forest, the properties of which depend primarily on the number, size and structure of the various glycosaminoglycan chains and their complex intermolecular affiliations.

The medical records, radiographs, and pathologic specimens of ten patients with the diagnoses of nontuberculous mycobacterial infection and acquired immunodeficiency syndrome (AIDS) were examined. The radiographic findings of alveolar or nodular infiltrates and adenopathy were relatively nonspecific but in most cases led to bronchoscopic study or open-lung biopsy, which established the diagnosis. Bronchoscopic washings or sputum cultures, which frequently provided the first confirmation of infection, were always followed by positive blood or tissue cultures. In contrast to nontuberculous infection in immunocompetent hosts, disseminated infection was common, with evidence of extrapulmonary involvement in nine patients. On the basis of these findings, we recommend that any AIDS patient with sputum or bronchoscopic washings demonstrating nontuberculous mycobacterial organisms be tentatively classified as having disseminated infection while being evaluated with blood, bone-marrow, stool, and urine cultures, even if the chest radiograph shows no disease.

Human colon carcinoma cells synthesize a high-molecular-weight heparan sulfate proteoglycan which is localized at the cell surface. In this study we have performed a series of immunoprecipitation and pulse-chase experiments associated with various pharmacological agents that interfere with the synthesis and post-translational modification of the proteoglycan. We demonstrate that colon carcinoma cells synthesize the heparan sulfate proteoglycan from a 400-kDa precursor protein that is immunologically related to the Engelbreth-Holm-Swarm (EHS) tumor cell proteoglycan. The cells contain a large pool of precursor protein with a half-life of about 75 min. Most of the precursor protein receives heparan sulfate side chains and is then transported to the cell surface and released into the medium. A portion of the precursor pool, however, does not receive heparan sulfate chains but is secreted into the medium. The glycosylation and subsequent secretion of the 400-kDa precursor protein was inhibited by NH4Cl and even more by monensin, indicating that the transit of precursor from the rough endoplasmic reticulum to the cell surface occurred through the Golgi complex and acidic compartments. The existence of a sizable pool of precursor protein was confirmed by additional experiments using cycloheximide and xyloside. These experiments showed that the half-life of the precursor protein was also 75 min and that stimulation of heparan sulfate synthesis by xyloside was greatly enhanced (about 12-fold) after new protein core synthesis was blocked by cycloheximide. Although the structural models proposed for the EHS and colon carcinoma heparan sulfate proteoglycans differ, the observation that they are derived from a precursor protein with dimensional and immunological similarities suggests that they may be genetically related.

Steroid secretion and structure of granulosa cells on floating collagen gels were compared with those of cells grown on plastic. Granulosa cells from follicles of gonadotropin-treated women were plated either onto dishes coated with type I collagen or onto plastic dishes. Medium containing serum was removed after 24 hours, defined medium was added, and the gel was floated. Medium was changed daily for 3 days, after which the granulosa cells were prepared for light and electron microscopy. Cells grown on collagen secreted significantly more estradiol and progesterone than those grown on plastic during the 3 days of culture. The round multilayered granulosa cells on collagen had abundant mitochondria and lipid droplets and they formed numerous intercellular junctions. On plastic surfaces, flat granulosa cells grew as a monolayer with few junctions and less abundant mitochondria or lipid droplets. We conclude that growth on floating collagen promotes structural changes of human granulosa cells that enhances cell interaction and secretion of steroid hormones.

Twenty dogs were studied to characterize the anatomic, hemodynamic and pathologic effects of catheter-delivered cardioversion (CDCV) in the left ventricle. Five dogs each received 1 CDCV of either 50, 100, 200 or 300 J at the left ventricular apex. The injury occurred at the posteroinferior apex. Structural integrity of the ventricle remained intact. Energy doses of 50 and 100 J resulted in focal subendomyocardial injury. Higher energy levels resulted in localized transmural injury. There was a linear dose-related correlation between the volume of injury and delivered energy. Electrocardiographic changes were seen immediately in all dogs. Fifteen dogs had acute, transient ventricular arrhythmias. One dog died with refractory arrhythmias. Minimal hemodynamic changes were associated with the CDCV. Unique histopathologic changes were observed. Thus, local tissue destruction can be produced with CDCV. The extent of injury is dose-related and is associated with minimum hemodynamic changes. The lesion may be acutely arrhythmogenic.

We report that keratin 8 (mK8) gene disruption causes colorectal hyperplasia in FVB/N mice. The intestinal lesions affect uniformly the cecum, colon, and rectum but not the small intestine. The elongation of the crypts is accompanied by an inflammation of the lamina propria and submucosa. Hepatic, renal, and pancreatic functions tested in clinical assays are within nonpathological range, suggesting that the major defect lies in colonic epithelial cells. Still, small but consistent elevation in the hepatic enzymes alanine (AST) and asparate (ALT) aminotransferase are observed, along with a 70% increase in spleen weight. No homozygous mouse line has been established, because of a markedly reduced fertility of the mK8-/- females. Previously, we reported that the mK8- targeted mutation causes embryonic lethality in (C57B1/6x129Sv) mice. This strong effect of the genetic background on the mK8- mutant phenotype emphasizes the importance of using several inbred mouse strains to reveal the polygenic contribution to mutant phenotypes. Our results demonstrate that genetic modifiers of K8/K18 filament functions, with profound effects on embryogenesis and gut functional integrity, are differentially active in the FVB/N and C57B1/6 genetic backgrounds. More importantly, the increase in mK8-/- gut epithelial cell number, rather than cell disruption, contrasts with the known function of epidermal keratins in providing mechanical strength.

Decorin is a well-known, ubiquitous proteoglycan that is a normal component of the ECM. Upon transgenic expression of decorin, tumor cells with diverse histogenetic background overexpress p21WAF1, a potent inhibitor of cyclin-dependent kinase activity, become arrested in G1, and fail to generate tumors in immunocompromised animals. Because decorin is a secreted protein, it has been recently suggested that decorin could act as an autocrine and paracrine regulator of tumor growth. Here, we demonstrate that adenovirus (Ad)-mediated transfer and expression of human decorin cDNA induced in vivo apoptosis of xenograft tumor cells in nude mice. This oncolytic activity was observed when the Ad vector encoding the decorin cDNA was injected intratumorally (i.t.) or i.v. Importantly, i.t. injection of the decorin Ad vector led to growth inhibition of the injected tumor associated with similar growth inhibition of a distant contralateral tumor, demonstrating a distant decorin antitumoral effect. Immunochemistry against human decorin and decorin quantitation in tumors confirmed that decorin migrated to the tumor distant site. Furthermore, decorin effect was specific to tumor cells, because neither apoptosis nor growth inhibition were observed in nontumoral human cells such as hepatocytes, endothelial cells, and fibroblasts, despite p21 overexpression. info:eu-repo/semantics/publishedVersion

// Shi-Qiong Xu 1, * , Simone Buraschi 2, * , Alaide Morcavallo 1, 3 , Marco Genua 1 , Tomoaki Shirao 4 , Stephen C. Peiper 2 , Leonard G. Gomella 1 , Ruth Birbe 2 , Antonino Belfiore 3 , Renato V. Iozzo 2 , Andrea Morrione 1 1 Department of Urology and Biology of Prostate Cancer Program, Thomas Jefferson University, Philadelphia, PA, USA 2 Department of Pathology, Anatomy and Cell Biology and Cancer Cell Biology and Signaling Program, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA, USA 3 Department of Health and Endocrinology, University Magna Graecia of Catanzaro, Catanzaro, Italy 4 Department of Neurobiology and Behavior, Gunma University School of Medicine, Showamachi, Maebashi, Japan * These authors have contributed equally to this work Correspondence to: Andrea Morrione, e-mail: Andrea.Morrione@jefferson.edu Renato V. Iozzo, e-mail: renato.iozzo@jefferson.edu Keywords: Progranulin, drebrin, bladder cancer, migration, invasion and anchorage-independent growth Received: October 13, 2014 Accepted: February 23, 2015 Published: March 12, 2015 ABSTRACT We recently established a critical role for the growth factor progranulin in bladder cancer insofar as progranulin promotes urothelial cancer cell motility and contributes, as an autocrine growth factor, to the transformed phenotype by modulating invasion and anchorage-independent growth. In addition, progranulin expression is upregulated in invasive bladder cancer tissues compared to normal controls. However, the molecular mechanisms of progranulin action in bladder cancer have not been fully elucidated. In this study, we searched for novel progranulin-interacting proteins using pull-down assays with recombinant progranulin and proteomics. We discovered that drebrin, an F-actin binding protein, bound progranulin in urothelial cancer cells. We characterized drebrin function in urothelial cancer cell lines and showed that drebrin is critical for progranulin-dependent activation of the Akt and MAPK pathways and modulates motility, invasion and anchorage-independent growth. In addition, drebrin regulates tumor formation in vivo and its expression is upregulated in bladder cancer tissues compared to normal tissue controls. Our data are translationally relevant as indicate that drebrin exerts an essential functional role in the regulation of progranulin action and may constitute a novel target for therapeutic intervention in bladder tumors. In addition, drebrin may serve as novel biomarker for bladder cancer.

Decorin, a small dermatan-sulfate proteoglycan, participates in extracellular matrix assembly and influences directly and indirectly cell behavior via interactions with signaling membrane receptors and transforming growth factor (TGF)-beta. We have therefore compared the development of tubulointerstitial kidney fibrosis in wild-type (WT) and decorin-/- mice in the model of unilateral ureteral obstruction. Without obstruction, kidneys from decorin-/- mice did not differ in any aspect from their WT counterparts. However, already 12 hours after obstruction decorin-/- animals showed lower levels of p27(KIP1) and soon thereafter a more pronounced up-regulation and activation of initiator and effector caspases followed by enhanced apoptosis of tubular epithelial cells. Later, a higher increase of TGF-beta1 became apparent. After 7 days, there was an up to 15-fold transient up-regulation of the related proteoglycan biglycan, which was mainly caused by the appearance of biglycan-expressing mononuclear cells. Other small proteoglycans showed no similar response. Because of enhanced degradation of type I collagen, end-stage kidneys from decorin-/- animals were more atrophic than WT kidneys. These data suggest that decorin exerts beneficial effects on tubulointerstitial fibrosis, primarily by influencing the expression of a key cyclin-dependent kinase inhibitor and by limiting the degree of apoptosis, mononuclear cell infiltration, tubular atrophy, and expression of TGF-beta1.

Epidermolysis bullosa (EB) in a group of genodermatoses characterized by the fragility of skin. Previous studies on the dystrophic (scarring) forms of EB have suggested abnormalities in anchoring fibrils, morphologically recognizable attachment structures that provide stability to the association of the cutaneous basement membrane to the underlying dermis. Since type VII collagen is the major component of the anchoring fibrils, we examined the genetic linkage of dominant dystrophic EB (EBDD) and the type VII collagen gene (COL7A1) locus, which we have recently mapped to chromosome 3p, in three large kindreds with abnormal anchoring fibrils. Strong genetic linkage of EBDD and COL7A1 loci was demonstrated with the maximum logarithm of odds (LOD) score of 8.77 at theta = 0. This linkage was further confirmed with two additional markers in this region of the short arm of chromosome 3, and these analyses allowed further refinement of the map locus of COL7A1. Since there were no recombinants between the COL7A1 and EBDD loci, our findings suggest that type VII collagen is the candidate gene that may harbor the mutations responsible for the EB phenotype in these three families.

A number of transmembrane proteins have been recently reported to be modified by the covalent addition of saturated fatty acids which may contribute to membrane targeting and specific protein-lipid interactions. Such modifications have not been reported in cell-associated heparan sulfate proteoglycans, although these macromolecules are known to be hydrophobic. Here, we report that a cell surface heparan sulfate proteoglycan is acylated with both myristate and palmitate, two long-chain saturated fatty acids. When colon carcinoma cells were labeled with [3H]myristic acid, a significant proportion of the label was shown to be specifically incorporated into the protein core of the proteoglycan. Characterization of fatty acyl moiety in the purified proteoglycan by reverse-phase high pressure liquid chromatography revealed that approximately 60% of the covalently bound fatty acids was myristate. We further show that this relatively rare 14-carbon fatty acid was bound to the protein core via a hydroxylamine- and alkali-resistant amide bond. The remaining 40% was the more common 16-carbon palmitate, which was bound via a hydroxylamine- and alkali-sensitive thioester bond. Palmitate appeared to be added post-translationally and derived in part from intracellular elongation of myristate, a process that occurred within the first two hours and was insensitive to inhibition of protein synthesis. Acylation of heparan sulfate proteoglycan represents a novel modification of this gene product and could play a role in a number of biological functions including specific interactions with membrane receptors and ligand stabilization.

The secreted proteoglycan decorin has been implicated in the negative control of cell proliferation primarily by virtue of its ability to block transforming growth factor-beta. Moreover, decorin expression is markedly up-regulated during quiescence but suppressed upon viral transformation, whereas de novo decorin expression in colon carcinoma cells abrogates the malignant phenotype by arresting the cells in the G1 phase of the cell cycle. Here we show that this decorin-induced growth arrest is associated with up-regulation of p21 mRNA and protein in a transforming growth factor-beta- and p53-independent pathway. The augmented p21 protein is present as a multimeric complex with various cyclins and cyclin-dependent kinases in the nuclei of decorin-expressing cells, thereby leading to suppression of cyclin-dependent kinase activity and block of cell division. Through the usage of decorin-specific antisense oligodeoxynucleotide treatment, we demonstrate that the expression of decorin is closely linked to that of p21 and that abrogation of decorin leads to suppression of p21 and restoration of cell division. Collectively, our results provide a plausible mechanism by which decorin may contribute to retard and suppress the growth of tumor cells in vivo.