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A novel sulfated glycosphingolipid that belongs to the "globo-series" was isolated from human kidney. This lipid was purified from a pooled kidney preparation by chloroform/methanol extraction, mild alkaline treatment, DEAE-Sephadex and silicic acid column chromatographies, and preparative thin layer chromatography. The structure and the properties were studied by infrared spectroscopy, two-dimensional proton magnetic resonance spectroscopy, negative secondary ion mass spectrometry, solvolysis, compositional and methylation analyses, monoclonal antibodies, and sulfatide-binding proteins. From the results of the above analyses, the structure of this glycolipid was proposed to be HSO3-3Gal beta 1-3GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc beta 1-1ceramide. The proton resonance at 3.93 ppm of the H-3 of the sulfated nonreducing terminal galactose of this lipid was downfield-shifted (delta 0.48 ppm), as compared with H-3 of the internal beta-galactose because of the electronegativity of the sulfate ester. This sulfated lipid reacted with a monoclonal anti-SSEA-3 (MC-631) (Kannagi, R., Cochran, N. A., Ishigami, F., Hakomori, S., Andrews, P. W., Knowles, B. B., and Solter, D. (1983) EMBO J. 2, 2355-2361), whose epitope is R-3GalNAc beta 1-3Gal alpha 1-4Gal beta 1-R', on thin layer chromatograms and solid-phase radioimmunoassay. This lipid also bound to the 125I-labeled sulfatide-binding protein, thrombospondin. The yield of this sulfated glycolipid was 0.19 nmol/g of tissue, which was about 0.09 and 0.5 mol % of galactosyl and lactosyl sulfatides in human kidney.

A single high-affinity binding site for adenine and related compounds was identified in the lima bean lectin (LBL) component III tetramer. This site is identical with the high affinity site for 2,6-toludinyl-naphthalenesulfonate described previously (Roberts, D. D., and Goldstein, I. J. (1982) J. Biol. Chem. 257, 11274-11277). [14C]Adenine was bound with high affinity (Kd = 1.2 +/- 0.1 X 10(-5) M, T = 25 degrees C) and a high degree of specificity in that hypoxanthine and guanine were very poor ligands for this site. Specificity was also observed for free purine bases relative to nucleosides or nucleotides. A number of N6 derivatives of adenine with cytokinin activity were found to bind to LBL, with relative affinities decreasing in the order: N6 - benzyladenine greater than kinetin greater than zeatin greater than N6 - [delta 2-isopentenyl]adenine greater than dihydrozeatin greater than zeatin riboside. Evidence was also obtained for heterotropic interaction between the adenine binding site and a second class of hydrophobic sites present on each subunit of LBL. Binding of adenine and N6-benzyladenine to LBL was found to produce a 2.3- and 3.8-fold increase, respectively, in the affinity of the lectin subunit hydrophobic sites for 1,8-anilinonaphthalenesulfonate. 1,8-Anilinonaphthalenesulfonate, in turn, enhanced the affinity of LBL for adenine, demonstrating that binding of ligands to the two classes of hydrophobic sites is thermodynamically linked. Equilibrium dialysis also revealed high affinity binding sites for [14C]adenine on the lectins from Dolichos biflorus, Phaseolus vulgaris, and soybean (Glycine max).

This report describes early experience with magnetic resonance imaging (MRI) of the temporomandibular joint meniscus in which surface coil technology was used. The results suggest remarkable imaging capabilities and speed with noninvasive methods.

A study of forty current business/technical/professional writing textbooks suggests that little disciplinary agreement exists about what proposals are and how they differ from some kinds of reports; how the various types of proposals should be classified; and what structural features characterize the genre. Though many texts blur the distinction between proposals and internal recommendation reports, the two are never the same. The textbooks present a bewildering array of classification systems, often failing to distinguish between situation and function. A function-based system could divide all proposals into two categories-analytic (research proposals, R&D proposals, and consulting proposals) and service/product, with bids representing a special case. The lack of disciplinary agreement also makes it difficult for textbook users to internalize a generic structure that will serve for all proposal-writing tasks. Such a structure would include the following: situation, objectives, methods, qualifications, costs, and benefits. The major advantages of such a generic structure are its slots, which make it like a schema; its event sequence, which makes it like a script; and its ability to help writers and teachers understand the relationship among the macropropositions that exist explicitly or implicitly in all proposals.

Recently developed computer programs create a new type of image from the sections created in computed tomography. These images look like actual photographs of internal structures. The authors describe the process of three-dimensional reconstruction in nonmathematical terms, and provide examples of its use in imaging the bones of the foot. They demonstrate the technique's ability to resolve small details, and its usefulness in displaying articular surfaces.

The challenge of fascism led Italy's most distinguished philosopher, Benedetto Croce, to focus on the condition of the philosophical foundations of liberalism. Partly as a result, Croce became a central figure in the liberal opposition to fascism, and when, in the early 1940's, it became possible to envision the reconstruction of a liberal political order in Italy, he had no choice but to play a major role, even though he was seventy-seven years old in 1943. Largely because of Croce's direct involvement, the ensuing debate over political priorities brought to the surface, to an unusual degree, major questions about the intellectual underpinnings of liberalism. Croce had been seeking to bring liberal theory up to date, with some promising results, but his immediate practical preoccupations during the 1940's diluted the force of his argument, so the contribution which he could have made to a modern recasting of liberalism, both for Italy and for modern culture in general, was not appreciated. Instead, Croce's stance contributed to tensions that quickly caused Italian liberalism to fragment — and to shrink — and of course the resulting weakness has been crucial to postwar Italian history. On the European level as well, the new liberalism he had in mind never fully coalesced, and thus in part, liberalism has been widely considered passé among European intellectuals.

Hydrophobic binding to the lectin from lima beans (Phaseolus lunatus) was studied by lectin-induced alterations in the fluorescence and absorption spectra of several hydrophobic ligands. The fluorescence of 1,8-anilinonaphthalenesulfonic acid (ANS) was greatly enhanced in the presence of lima bean lectin with concomitant shift of the fluorescence emission maximum from 520 to 469 nm. Similar enhancement was seen with 2,6-toluidinylnaphthalenesulfonic acid (TNS) with a shift of emission from 500 to 423 nm, and with an uncharged analogue, N-phenyl-1-naphthylamine. Fluorescence titrations with ANS and rose bengal yielded affinity constants of 3.9 X 10(3) and 6 X 10(5) M-1, respectively. Fluorescence titration with TNS indicated binding heterogeneity and yielded intrinsic association constants of 7.8 X 10(4) and 2.2 X 10(3) M-1 assuming a model with two classes of independent sites. The high affinity binding had an apparent stoichiometry of 1.08 sites/lectin tetramer. Equilibrium dialysis for ANS and TNS confirmed the results of fluorescence titration and gave stoichiometries for ANS and low affinity TNS binding of one site/subunit. Neither chemical modification of thiol groups known to be essential for carbohydrate binding nor titration in the presence of haptenic sugar affected the binding of ANS or TNS to the lectin. These results indicated that the carbohydrate and hydrophobic binding sites of lima bean lectin are independent and noninteracting.

The dependence on thiol pK of the second-order rate constant (kS) for reaction of thiolate anions with MMTS was shown to follow the Brønsted equation log kS = log G + beta pK with log G = 1.44 and 3.54 and beta = 0.635 and 0.309 for aryl and alkyl thiols, respectively. The reactivity toward MMTS of the protonated thiol group was found to be negligible in comparison to that of the thiolate anion. For 2-mercaptoethanol the reactivity toward MMTS of the protonated form of the thiol group was shown to be at least 5 X 10(9) smaller than that of the thiolate anion. The pH dependence of the second-order rate constant for reaction of the thiolate group of Cys-25 at the active site of papain was determined and shown to be consistent with the previously determined low pK for Cys-25 and its electrostatic interaction with His-159. The small dependence of the reactivity of Cys-25 on thiol pK (beta approximately 0.09) suggested that the charge-charge interactions that act through space to perturb the pK of the nucleophile at the active site of papain and perhaps other enzymes may serve to increase the fraction of nucleophile present in the reactive basic form without introducing the decrease in nucleophilic reactivity seen in model systems where pK's are lowered primarily by charge-dipole interactions.

An N-acetyl-D-galactosamine-specific lectin has been isolated from the two seed forms of the hog peanut (Amphicarpaea bracteata) using an affinity support containing the synthetic type A blood group trisaccharide alpha-D-GalNAc-(1,3)-[alpha-L-Fuc-(1,2)]-beta-D-Gal (Synsorb A). The affinity-purified lectin appears to be identical in both seed types. Gel filtration on Sephadex G-200 gives a single symmetrical peak corresponding to Mr 135,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows four subunit forms, each of which contains carbohydrate. Limited amino terminal sequencing indicates heterogeneity in two of the first 10 residues. The lectin contains no cysteine. There are four equivalent, noninteracting GalNAc binding sites per 135,000-Da molecule, having an association constant for methyl N-acetyl-alpha-D-galactosaminide of 4.0 X 10(4) M-1. Precipitin and hapten inhibition studies show the lectin to be specific for terminal, nonreducing D-GalNAc units, with a preference for the alpha-anomer and enhanced specificity for the disaccharide, GalNAc alpha 1,3GalNAc. There is also a single adenine binding site per Mr 135,000 lectin molecule with an association constant of 1.3 X 10(6) M-1.

Pseudomonas aeruginosa infection in the lungs is a leading cause of death of patients with cystic fibrosis, yet a specific receptor that mediates adhesion of the bacteria to host tissue has not been identified. To examine the possible role of carbohydrates for bacterial adhesion, two species of Pseudomonas isolated from patients with cystic fibrosis were studied for binding to glycolipids. P. aeruginosa and P. cepacia labeled with 125I were layered on thin-layer chromatograms of separated glycolipids and bound bacteria were detected by autoradiography. Both isolates bound specifically to asialo GM1 (Gal beta 1-3GalNAc beta 1-4Gal beta 1-4Glc beta 1-1Cer) and asialo GM2 (GalNAc beta 1-4Gal beta 1-4Glc beta 1-1Cer) but not to lactosylceramide (Gal beta 1-4Glc beta 1-1Cer), globoside (GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc beta 1-1Cer), paragloboside (Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1Cer), or several other glycolipids that were tested. Asialo GM1 and asialo GM2 bound the bacteria equally well, exhibiting similar binding curves in solid-phase binding assays with a detection limit of 200 ng of either glycolipid. Both isolates also did not bind to GM1, GM2, or GDla suggesting that substitution of the glycolipids with sialosyl residues prevents binding. As the Pseudomonas do not bind to lactosylceramide, the beta-N-acetylgalactosamine residue, positioned internally in asialo GM1 and terminally in asialo GM2, is probably required for binding. beta-N-Acetylgalactosamine itself, however, is not sufficient as the bacteria do not bind to globoside or to the Forssman glycolipid. These data suggest that P. aeruginosa and P. cepacia recognize at least terminal or internal GalNAc beta 1-4Gal sequences in glycolipids which may be receptors for these pathogenic bacteria.

Despite recent advances in diagnostic radiology, current techniques for radiographic evaluation of the temporomandibular joint continue to present the clinician with difficult problems in interpretation and diagnosis. The use of three-dimensional images reconstructed from computed tomographic (CT) data improves the diagnostic value of conventional CT at no additional risk to the patient and may provide new insights into this complex anatomic structure. A sequence of 1.5 mm CT sections is made with a slice-to-slice spacing of 1.0 mm. These are used to construct a new sequence of slices by mathematical interpolation in which the new slice spacing equals the size of the pixels. Structures to be imaged separately are then masked in the interpolated sections prior to “windowing” for the appropriate tissue density. A special algorithm detects the boundary surface of the selected structure. The surface pixels are assigned gray levels on the basis of their distance and attitude from the observer. When displayed, this produces a simulated three-dimensional image. The image can be rotated and sectioned. Rotations permit otherwise hidden surfaces to be examined. Images of a human temporomandibular joint in vitro are presented to demonstrate (1) the bony components of the joint and their relationships within the joint; (2) the bony components separated to display hidden surfaces; and (3) the joint meniscus in situ and as a separate component.

Binding of the Hydrophobic ligands 1,8-anilinonaphthalenesulfonic acid (ANS) and 2,6-toluidinylnaphthalenesulfonic acid (TNS) to a variety of plant lectins was studied by lectin-induced alteration of the fluorescence spectra of the two ligands. With one exception, all legume lectins examined bound ANS, with affinity constants ranging from 103 to 104 M−1. Similar ANS binding was noted for some nonlegume lectins. Titration of the five isolectins from Phaseolus vulgaris with ANS indicated positive cooperative binding of ANS to the two isolectins E4 and E3L1. Titrations with TNS revealed high-affinity sites for this ligand in a number of lectins. Addition of haptenic sugars did not inhibit binding of ANS, suggesting that the hydrophobic binding sites of lectins are independent of the carbohydrate binding sites.

The adhesive glycoproteins laminin, thrombospondin and von Willebrand's factor bind specifically and with high affinity to sulfated glycolipids, and it is this binding that probably accounts for their ability to agglutinate glutaraldehyde-fixed erythrocytes. The 3 proteins differ, however, in the effect of sulfated polysaccharides on their binding to sulfatides. Fucoidan strongly inhibits binding of both laminin and thrombospondin, but not of von Willebrand's factor, suggesting the involvement of laminin or thrombospondin or other unknown sulfatide-binding proteins in specific cell interactions that are also inhibited by fucoidan. Thrombospondin adsorbed onto plastic promotes the attachment and spreading of G361 melanoma cells. Interestingly, fucoidan and an antibody directed against the sulfatide-binding domain of thrombospondin selectively inhibit spreading but not attachment. Sulfatides, but not neutral glycolipids or gangliosides, when adsorbed onto plastic also promote attachment and spreading of G361 melanoma cells. Direct adhesion of G361 cells requires high densities of sulfatide. In the presence of laminin, however, specific adhesion of G361 cells to sulfatide is strongly stimulated and requires only low densities of adsorbed lipid, suggesting that laminin mediates adhesion by cross-linking receptors on the melanoma cell surface to sulfatide adsorbed onto the plastic. Although thrombospondin binds to sulfatide and to G361 cells, it does not enhance but rather inhibits direct and laminin-dependent G361 cell adhesion to sulfatide, presumably because it is unable to bind simultaneously to ligands on opposing surfaces. Thus, sulfated glycoconjugates participate in both laminin- and thrombospondin-mediated cell adhesion, but their mechanisms of interaction are different.

Publisher Summary Cell surface glycoconjugates have been extensively studied as potential receptors for cell–cell and cell–ligand interactions. Carbohydrates occur on cell surface glycoproteins, glycolipids, and proteoglycans. As glycolipids containing a single oligosaccharide are readily purified and terminal structures on glycolipids are often identical to those found on glycoproteins, glycolipids are useful for examining the specificity of proteins which may bind to cell surface carbohydrates. A second class of acidic glycolipids contains sulfate esters. These lipids are found in many tissues and occur with various carbohydrate sequences. Most contain sulfate esters of galactose, but sulfation also occurs on N -acetylgalactosamine, N -acetylglucosamine, glucose, and sialic acid. Several methods have been used to study sulfatide-binding proteins. Enzymatic activation and changes in fluorescence polarization of factor XII and kininogen occur in the presence of sulfatide micelles. Numerous methods for the preparation of lipid extracts from tissues are effective for sulfated glycolipids. This chapter presents a generalized approach for characterizing sulfatide-binding proteins using immobilized sulfatides and outlines the criteria for defining the specificity of these interactions.

The carbohydrate binding stoichiometry of lima bean lectin component III was reexamined using equilibrium dialysis and quantitative affinity chromatography following limited chemical modification. Equilibrium dialysis employing methyl[2- 14 C]benzamido-2-deoxy-α- d -galactopyranoside as ligand demonstrated that the lectin tetramer bound 4 mol of sugar with K assoc = 1.44 ± 0.13 × 10 3 m −1 (T = 5 °C, pH 7.0, ionic strength 0.1). The previous report of two sites/tetramer [Bessler, W. and Goldstein, I. J. (1974) Arch. Biochem. Biophys. 165 , 444] appears to be the result of partial inactivation of the lectin due to oxidation of essential thiol groups. Following limited chemical modification of the thiol groups by methyl methanethiosulfonate, multiple intermediate forms with reduced affinity for Synsorb A were obtained. The number and hemagglutinating activities of these intermediates provided further support for the presence of four carbohydrate binding sites on lima bean lectin component III.

We investigated whether thrombospondin plays a role in the binding of Plasmodium falciparum parasitized erythrocytes to C32 melanoma cells. Twelve patient isolates bound variably to melanoma cells, with good correlation between the degree of binding to cells and binding to thrombospondin. With a synchronous preparation of asexual parasites, acquisition of the capacity to bind to thrombospondin occurred at the same parasite stage as binding to melanoma cells. Development of parasites to trophozoites and schizonts correlated with binding of parasitized erythrocytes to thrombospondin and melanoma cells. The infected erythrocyte receptor for thrombospondin was destroyed by mild trypsinization, as was the receptor for melanoma cells. Although these results suggest similarity in the melanoma cell receptor and thrombospondin receptor for infected cells, other results showed that thrombospondin cannot alone be the melanoma cell receptor. Binding to other melanoma cell lines did not correlate with thrombospondin secretion: the RPMI 8252 and G361 cell lines bound few or no infected cells, yet secreted 50-100% as much thrombospondin as C32 cells. Iodinated thrombospondin bound in similar amounts to C32 cells and to noncytoadherent C361 melanoma cells. Binding and nonbinding melanoma cells did not differ in quantity of surface thrombospondin by radioimmunoassay. Thus, although purified, immobilized, thrombospondin binds parasitized erythrocytes, expression of thrombospondin alone on melanoma cells is not sufficient to mediate adherence.

Computerized tomography has some limitations for diagnosis of internal derangements of the TMJ. The use of three-dimensional image reconstructions of CT data may enhance the diagnostic utility of CT. In a cadaver simulation, three-dimensional imaging was able to demonstrate the location of a displaced meniscus which was not obvious in two-dimensional sections. Location of the meniscus was aided by the use of a "transparency mode" in displaying the data.

The combining site of the lima bean (Phaseolus lunatus) lectin (LBL) was studied by quantitative precipitin and precipitin-inhibition assays. The lectin precipitated best with hog gastric mucosa and human ovarian cyst blood group A1 substances and moderately with A2 substances. B substances precipitated very poorly and H, Lea, Leb, and precursor I substances did not react. Blood group A1 and A2 substances reacted to varying extents and these differences are attributable to heterogeneity resulting from incomplete biosynthesis of carbohydrate chains. By inhibition of precipitation of LBL with A1 blood group substance, the lectin was found to be most specific for fucose-containing oligosaccharides having the A trisaccharide, DGalNAc alpha 1----3[L-Fuc alpha 1----2]DGal determinant. The best inhibitor, an A-specific hexasaccharide, DGalNAc alpha 1----3[LFuc alpha 1----2]DGal beta 1----3DGlcNAc beta 1----3-DGal beta 1----4DGlc, was 11 times more active than the A trisaccharide. A difucosyl oligosaccharide with a second fucose linked alpha 1----3 to the DGlcNAc is less active; fucose linked alpha 1----4 to DGlcNAc was completely inactive. These results suggest that specific interactions with the subterminal sugars may be important in the binding, and that the specificity of the lectin combining site involves at least the nonreducing terminal four and probably five sugars of the hexasaccharide. Thus LBL has a more-extended binding site than was inferred previously and is in the upper range of antibody combining-site sizes.

Interpretational difficulties experienced with currently used diagnostic radiation techniques can be reduced via the use of 3-D images constructed from conventional CT data. Each 1.5 mm CT section yields interpolated sections (6 or 8) containing cubile voxels. Structures to be imaged separately are masked in the interpolated sections prior to windowing for the appropriate tissue. A special algorithm detects the surface boundary of the selected structure. The surface pixels are assigned gray levels based on their distance and attitude from the observer. When displayed, this produces a simulated 3-D image. The image can be rotated and sectioned. Rotations permit otherwise hidden surfaces to be examined. Images of two temporomandibular joints are presented, a) bony components; b) bony components + meniscus. It is concluded that the 3-D imaging process is potentially useful in diagnosing TMJ pathology.

Monoclonal antibody 19-9 detects a sialosylated Lea antigen with the following sugar sequence: NeuNAc alpha 2-3Gal beta 1-3[Fuc alpha 1-4]GlcNAc beta 1-3Gal. . . . This antigen is detected as a mucin in the sera of many patients with gastrointestinal and pancreatic cancer. Elevated levels of sialosylated Lea antigen are also detected in serum from 14 of 16 patients with cystic fibrosis (87%). One of the two negative patients belongs to the Le(a-b-) blood group and so is unable to synthesize the sialosylated Lea antigen. The high percentage of cystic fibrosis patients with elevated sialosylated Lea antigen suggests that the 19-9 antibody may be useful for diagnosis of cystic fibrosis. Antibodies to other sialosylated carbohydrates in mucins may also be useful for detection of cystic fibrosis and may allow diagnosis of patients belonging to the Le(a-b-) blood group.

Publisher Summary The anti-A seed lectin from Amphicarpaea bracteata is one of several Leguminosae lectins specific for terminal nonreducing N-acetyl-D-galactosamine (D-GalNAc)2 groups. The lectin, originally purified by affinity chromatography on N-acetyl-D-galactosamine coupled to epoxy-Sepharose 6B, was reported to be homogeneous by sodium dodecyl sulfate (SDS)-polyacrylamide electrophoresis, and an initial characterization was reported. This chapter describes a new purification procedure and further characterization of the physical and chemical properties of the A. bracteata lectin. The assay methods used on this lectin are hemagglutination assays and quantitative precipitation inhibition assay. The chapter describes the purification process of the lectin in detail, which includes extraction and affinity chromatography. There is also some discussion about the properties of the lectin from Amphicarpaea bracteata. A. bracteata lectin falls in a class of several plant lectins that contain an adenine-binding site in addition to the carbohydrate site.

The asparagine-linked sugar chains of human platelet thrombospondin were released as oligosaccharides by hydrazinolysis. About 12 mol of sugar chains was released from one thrombospondin molecule. This was converted to radioactive oligosaccharides by sodium borotritide reduction after N-acetylation, and separated into one neutral and four acidic fractions by paper electrophoresis. More than 90% of the oligosaccharides were recovered in the acidic fraction. The acidic oligosaccharides were mostly converted to neutral oligosaccharides by sialidase treatment, indicating that they are sialyl derivatives. The neutral and sialidase-treated acidic oligosaccharides were further fractionated by Bio-Gel P-4 column chromatography. Structural study of each oligosaccharide by sequential exoglycosidase digestion and methylation analysis revealed that the thrombospondin contains mono-, bi-, tri-, and tetraantennary complex-type sugar chains in addition to a small amount of high-mannose type. Approximately 70% of the complex-type sugar chains was fucosylated at asparagine-linked N-acetylglucosamine residue and 19% of the biantennary complex-type sugar chains was bisected.

CD47 is a signaling receptor for thrombospondin-1 and the counter-receptor for signal-regulatory protein-α (SIRPα). By inducing inhibitory SIRPα signaling, elevated CD47 expression by some cancers prevents macrophage phagocytosis. The anti-human CD47 antibody B6H12 inhibits tumor growth in several xenograft models, presumably by preventing SIRPα engagement. However, CD47 signaling in nontransformed and some malignant cells regulates self-renewal, suggesting that CD47 antibodies may therapeutically target cancer stem cells (CSCs). Treatment of MDA-MB-231 breast CSCs with B6H12 decreased proliferation and asymmetric cell division. Similar effects were observed in T47D CSCs but not in MCF7 breast carcinoma or MCF10A breast epithelial cells. Gene expression analysis in breast CSCs treated with B6H12 showed decreased expression of epidermal growth factor receptor (EGFR) and the stem cell transcription factor KLF4. EGFR and KLF4 mRNAs are known targets of microRNA-7, and B6H12 treatment correspondingly enhanced microRNA-7 expression in breast CSCs. B6H12 treatment also acutely inhibited EGF-induced EGFR tyrosine phosphorylation. Expression of B6H12-responsive genes correlated with CD47 mRNA expression in human breast cancers, suggesting that the CD47 signaling pathways identified in breast CSCs are functional in vivo. These data reveal a novel SIRPα-independent mechanism by which therapeutic CD47 antibodies could control tumor growth by autonomously forcing differentiation of CSC.

Magnetic resonance (MR) imaging of the temporomandibular joint was performed in two subjects using a 1.5 T experimental imaging system equipped with a 6.5 cm surface coil antenna. Normal and pathologic anatomy were demonstrated with exquisite detail. Anterior displacement of the joint meniscus was clearly visible in the symptomatic subject, consistent with arthrographic confirmation.

MUCH has been written about the theories and principles of Tory paternalism in England, but little has been said about its practice. Samuel Taylor Coleridge in his later writings gave eloquent and profound expression to its principles; Robert Southey in his Colloquies on the Progress of Society espoused them with warmth and imagination. In i838 William Gladstone, still a very earnest Tory, reaffirmed the ecclesiastical ideals, of Coleridge in his famous discussion of The State in Its Relations with the Church. Twelve years later Thomas Carlyle declared war against liberalism by writing his Latter Day Pamphlets, in which he fulminated against those who worship mammon. Benjamin Disraeli in his novels Sybil and Coningsby dressed Tory paternalism in a gallant and chivalrous costume and taught more than one generation to admire its lofty sentiments. The ideas of these writers, varied and complex as they are, have caught the imagination of a distinguished array of historians and political philosophers. Alfred Cobban, Crane Brinton, and R. J. White have written brilliantly, and at times sympathetically, about them. But it has remained for the new conservatives of our age to write about them in a spirit of hallowed veneration. Anxious to construct a political philosophy to clothe the present conservative temper of America, they have looked back to the humanitarian, Christian, and romantic philosophy of Coleridge and Southey. Clinton Rossiter in Conservatism in America expresses his admiration for Coleridge and Disraeli. Peter Viereck in Conservatism Revisited praises that English conservatism which "tends to put social justice ahead of laissez faire." Neither of them, however, gives such praise to Tory paternalism as does Russell Kirk. In The Conservative Mind, Kirk draws once again the classic picture of Tory paternalism pitted against an unsentimental liberalism, a picture familiar to all who have read Monypenny and Buckle's Life of Benjamin Disraeli, or Wingfield-Stratford's History of British Civilization, or even Keith Feiling's recent History of England. In redrawing this picture Kirk has added color; it now has its heroes and its villains, contesting for the souls of men and struggling to control the destinies of the new industrial society. The backdrop to this drama is a scene of grim factory towns with overworked

Background The secreted enzyme autotaxin (ATX) stimulates tumor cell migration, tumorigenesis, angiogenesis, and metastasis. ATX hydrolyzes nucleotides, but its hydrolysis of lysophospholipids to produce lysophosphatidic acid (LPA) accounts for its biological activities. ATX has been identified only as a constitutively active enzyme, and regulation of its activity is largely unexplored. In spite of its presence in plasma along with abundant putative substrate LPC, the product LPA is found in plasma at unexpectedly low concentrations. It is plausible that the LPA-producing activity of ATX is regulated by its expression and by access to substrate(s). For this reason studying the interaction of enzyme with substrate is paramount to understanding the regulation of LPA production. Results In this study we determine ATX hydrolytic activities toward several artificial and natural substrates. Two novel point mutations near the enzyme active site (H226Q and H434Q) confer attenuated activity toward all substrates tested. The Vmax for LPC compounds depends upon chain length and saturation; but this order does not differ among wild type and mutants. However the mutant forms show disproportionately low activity toward two artificial substrates, pNpTMP and FS-3. The mutant forms did not significantly stimulate migration responses at concentrations that produced a maximum response for WT-ATX, but this defect could be rescued by inclusion of exogenous LPC. Conclusion H226Q-ATX and H434Q-ATX are the first point mutations of ATX/NPP2 demonstrated to differentially impair substrate hydrolysis, with hydrolysis of artificial substrates being disproportionately lower than that of LPC. This implies that H226 and H434 are important for substrate interaction. Assays that rely on hydrolyses of artificial substrates (FS-3 and pNpTMP), or that rely on hydrolysis of cell-derived substrate, might fail to detect certain mutated forms of ATX that are nonetheless capable of producing LPA in the presence of sufficient exogenous substrate. H420Q-ATX could not be differentiated from WT-ATX, indicating that histidine at position 420 is not required for any of the activities of ATX tested in this study.

Background Soluble guanylate cyclase (sGC) is the signal transduction enzyme most responsible for mediating the effects of nitric oxide by producing cyclic guanosine monophosphate (cGMP) from guanosine triphosphate (GTP). NOcGMP signaling is vital to proper cardiovascular function. Deficiencies in the production of either NO or cGMP contribute to cardiovascular disorders. Therapies designed to increase in vivo NO levels (such as organic nitrates) have well documented clinical limitations due to drug tolerance. Recently, a variety of NO-independent small molecule activators of sGC have been reported that activate nearly or as well as NO and have promising clinical activities. Previous results from our lab have shown that the secreted matrix protein thrombospondin-1 (TSP-1) binds to CD47 and potently inhibits NO stimulation of sGC.

Background Autophagy (macroautophagy), a cellular process of “self-eating”, segregates damaged/aged organelles into vesicles, fuses with lysosomes, and enables recycling of the digested materials. The precise origin(s) of the autophagosome membrane is unclear and remains a critical but unanswered question. Endoplasmic reticulum, mitochondria, Golgi complex, and the plasma membrane have been proposed as the source of autophagosomal membranes. Findings Using electron microscopy, immunogold labeling techniques, confocal microscopy, and flow cytometry we show that mitochondria can directly donate their membrane material to form autophagosomes. We expand upon earlier studies to show that mitochondria donate their membranes to form autophagosomes during basal and drug-induced autophagy. Moreover, electron microscopy and immunogold labeling studies show the first physical evidence of mitochondria forming continuous structures with LC3-labeled autophagosomes. The mitochondria forming these structures also stain positive for parkin, indicating that these mitochondrial-formed autophagosomes represent a novel mechanism of parkin-associated mitophagy. Conclusions With the on-going debate regarding autophagosomal membrane origin, this report demonstrates that mitochondria can donate membrane materials to form autophagosomes. These structures may also represent a novel form of mitophagy where the mitochondria contribute to the formation of autophagosomes. This novel form of parkin-associated mitophagy may be a more efficient bio-energetic process compared with de novo biosynthesis of a new membrane, particularly if the membrane is obtained, at least partly, from the organelle being targeted for later degradation in the mature autolysosome.