The shape of the triangular fibrocartilage during pronation-supination.

We studied the changes in the shape of the triangular fibrocartilage (TFC: disc proper) which occur during forearm rotation in disarticulated and articulated wrists. The influence of artificial 3mm ulnar lengthening on distortion of the disc was also examined. In the disarticulated wrists, slight distortion of the central and radial portions of the TFC was observed in the ulnar neutral variance specimens. More distortion was noted in the radial and central portions of the TFC in specimens with positive ulnar variance or with the ulna lengthened. However, in the articulated wrist, the TFC demonstrated little change in shape during pronosupination even in the ulnar positive variance wrists or with the ulna lengthened. There was no significant change in palmar and dorsal peripheral lengths of the TFC in ulnar neutral, ulnar positive or ulna-lengthened specimens at three rotatory positions of the forearm. These findings suggest that changes in ulnar variance which occur during forearm rotation can produce distortion on the TFC, but the carpus helps to maintain the shape of the TFC during pronation-supination, even with positive ulnar variance.

The proximal ligamentous component of the triangular fibrocartilage complex.

The proximal ligamentous component of the triangular fibrocartilage complex (TFCC) was studied anatomically using 15 fresh-frozen cadaver hand forearm specimens. Changes in the length of either side of this component were analysed during forearm rotation with the complete three-dimensional structure of the TFCC preserved. The proximal ligamentous component consists of three portions: dorsal, central and palmar. The dorsal and palmar portions connect the radius and ulna directly. These were recognized in all specimens whereas the central portion was not constant. The morphology of the proximal component was categorized into three types: fan-shaped, V-shaped, and funnel-shaped in five wrists each. Changes in ligament length during forearm rotation were measured using fine wires under slight tension that paralleled the ligaments from origin to insertion. The dorsal and palmar portions demonstrated three trends: the dorsal portion increased in length from supination to pronation whereas the palmar portion increased in length from pronation to supination; the length of the dorsal portion remained almost constant as the palmar portion increased in length from pronation to supination; the length of the palmar portion remained almost constant while the dorsal portion lengthened from supination to pronation. These variations appear to be related to which portion of the ligament was attached nearest to the centre of the ulnar fovea, where the rotational axis of the forearm passes. The portion attaching nearest to the fovea demonstrated a nearly isometric length pattern, whereas the portion which attached at a distance showed greater extensibility. These findings suggest that the proximal component of the TFCC corresponds to a true radioulnar ligament, and the isometric and eccentric fibres act mutually during forearm rotation.

Involvement of Ras in the expression of glycolipid sulfotransferase in human renal cancer cells.

Glycolipid sulfotransferase activity in a human renal cell carcinoma cell line, SMKT-R3, is enhanced by epidermal growth factor (EGF); tyrosine kinase inhibitors suppress this enhancement. To investigate the involvement of Ras in the signal transduction pathway from the EGF receptor to the expression of glycolipid sulfotransferase, we introduced v-H-ras into SMKT-R3 cells. In a quiescent state, the percent GTP bound to Ras in v-H-ras-expressing cells increased about 2.5-fold compared with control cells, suggesting that v-Ras introduced into the renal cancer cells is in an active form without EGF stimulation. Glycolipid sulfotransferase activity in v-H-ras-expressing cells was higher than in control cells. The sulfotransferase activity was affected neither by EGF nor by genistein, a tyrosine kinase inhibitor, in v-H-ras-expressing cells, whereas it was enhanced by EGF and reduced by genistein in control cells. Our observations suggest that Ras mediates the regulation pathway of glycolipid sulfotransferase activity in SMKT-R3 cells.

Molecular cloning and expression of cDNA encoding human 3'-phosphoadenylylsulfate:galactosylceramide 3'-sulfotransferase.

We have isolated a cDNA clone encoding human 3'-phosphoadenylylsulfate:galactosylceramide 3'-sulfotransferase (EC 2.8.2.11). Degenerate oligonucleotides, based on amino acid sequence data for the purified enzyme, were used as primers to amplify fragments of the gene from human renal cancer cell cDNA by the polymerase chain reaction method. The amplified cDNA fragment was then used as probe to screen a human renal cancer cell cDNA library. The isolated cDNA clone contained an open reading frame encoding 423 amino acids including all of the peptides that were sequenced. The deduced amino acid sequence predicts a type II transmembrane topology and contains two potential N-glycosylation sites. There is no significant homology between this sequence and either the sulfotransferases cloned to date or other known proteins. Northern blot analysis demonstrated that a 1.9-kilobase mRNA was unique to renal cancer cells. When the cDNA was inserted into the expression vector pSVK3 and transfected into COS-1 cells, galactosylceramide sulfotransferase activity in the transfected cells increased from 8- to 16-fold over that of controls, and the enzyme product, sulfatide, was expressed on the transformed cells.

Purification and characterization of 3'-phosphoadenosine-5'-phosphosulfate:GalCer sulfotransferase from human renal cancer cells.

We have purified 3'-phosphoadenosine-5'-phosphosulfate:GalCer sulfotransferase [EC 2.8.2.11] from a human renal cancer cell line SMKT-R3 through a combination of affinity chromatographies using galactosylsphingosine, 3',5'-bisphosphoadenosine and heparin as ligands. The purified sulfotransferase showed a specific activity of 1.2 mumol/min/mg, which is 300 times more than the highest activity among the enzyme preparations purified so far from other sources. Homogeneity of the purified sulfotransferase was supported by the facts that the enzyme preparation showed a single protein band with an apparent molecular mass of 54 kDa on reducing SDS-PAGE and that protein bands coincided with the enzyme activity on both native PAGE and nonreducing SDS-PAGE. GalCer was the best acceptor for the purified enzyme. LacCer, GalAAG, and GalDG were also good acceptors. GlcCer, Gg3Cer, Gg4Cer, Gb4Cer, and nLc4Cer did serve as acceptors although the relative activities were low. On the other hand, the enzyme could not act on Gb3Cer, which possesses alpha-galactoside at the nonreducing terminus. Neither galactose nor lactose served as an acceptor. These observations suggest that the sulfotransferase prefers beta-glycoside, especially beta-galactoside, at the nonreducing termini of sugar chains attached to a lipid moiety.

Regulation of glycolipid sulfotransferase by tyrosine kinases in human renal cancer cells.

Glycolipid sulfotransferase activity in a human renal cancer cell line, SMKT-R3, is enhanced by the action of growth factors such as EGF, TGF-alpha and HGF, whose receptors possess tyrosine kinase domains. We investigated whether tyrosine kinases are involved in the regulation of the sulfotransferase in the cells by using specific tyrosine kinase inhibitors. Genistein and tyrphostin 51 not only cancelled the enhancement of the sulfotransferase by EGF but also reduced the enzyme level to a point much lower than that seen in non-treated cells, whereas they did not affect the sulfotransferase activity in vitro. The activity-reducing effects of genistein were dose- and time-dependent. Genistein also inhibited the cell growth of SMKT-R3 cells. Western blotting using anti-phosphotyrosine monoclonal antibody revealed a tyrosine-phosphorylated protein with an apparent molecular mass of 116 kDa in the non-treated cells. The EGF receptor was tyrosine-phosphorylated by the addition of EGF. The phosphorylations of the 116 kDa protein and EGF receptor were attenuated by co-incubation with genistein. These results indicate that tyrosine kinases including the EGF receptor are involved in the growth of SMKT-R3 cells and in the regulatory mechanisms of glycolipid sulfotransferase in the cells.

Pyridoxal 5'-phosphate binds to a lysine residue in the adenosine 3'-phosphate 5'-phosphosulfate recognition site of glycolipid sulfotransferase from human renal cancer cells.

In the course of characterization of glycolipid sulfotransferase from human renal cancer cells, the manner of inhibition of sulfotransferase activity with pyridoxal 5'-phosphate was investigated. Incubation of a partially purified sulfotransferase preparation with pyridoxal 5'-phosphate followed by reduction with NaBH4 resulted in an irreversible inactivation of the enzyme. When adenosine 3'-phosphate 5'-phosphosulfate was coincubated with pyridoxal 5'-phosphate, the enzyme was protected against this inactivation. Furthermore, pyridoxal 5'-phosphate was found to behave as a competitive inhibitor with respect to adenosine 3'-phosphate 5'-phosphosulfate with a Ki value of 287 microM. These results suggest that pyridoxal 5'-phosphate modified a lysine residue in the adenosine 3'-phosphate 5'-phosphosulfate-recognizing site of the sulfotransferase.

Enzymatic sulfation of gangliotriaosylceramide in human renal cancer cells.

Biosynthesis of sulfoglycolipid is markedly increased in a human renal cancer cell line, SMKT-R3. We investigated the sulfotransferase catalyzing the transfer of sulfate from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to gangliotriaosylceramide (Gg3Cer) in SMKT-R3 cells. On thin-layer chromatography, the reaction product comigrated with Gg3Cer III3-sulfate (SM2b), not with Gg3Cer II3-sulfate (SM2a). To examine which monosaccharide of Gg3Cer was sulfated, the product was treated with beta-hexosaminidase A. Unlike authentic SM2a, of which the non-reducing terminal N-acetylgalactosamine was cleaved off, the product was not hydrolyzed. These results suggest that sulfate was transferred to the non-reducing terminal N-acetylgalactosamine. Gg3Cer sulfotransferase activity was independent of divalent cations but stimulated by Fe2+, and the optimal pH was approximately 6.5. The apparent Km values were 102 microM for PAPS and 348 microM for Gg3Cer. Gg3Cer II3,III3-bis-sulfate (SB2) and a sulfotransferase activity synthesizing SB2 from SM2a were also detected in SMKT-R3 cells.

Zn-alpha 2-glycoprotein is a novel adhesive protein.

An amino acid sequence (Arg-Gly-Asp-Val) specifically associating with cell adhesion between cells and extracellular matrices was found on the human Zn-alpha 2-glycoprotein (Zn alpha 2gp) molecule. Although other mammalian cell lines such as breast carcinoma and melanoma did not, SMKT R-3 cells (human renal cell carcinoma) but not the kidney cell lines (Vero and COS7) preferentially attached and spread on a tissue culture plate coated with either blood plasma Zn alpha 2gp or seminal plasma Zn alpha 2gp. The spreading of SMKT R-3 cells on Zn alpha 2gp required divalent cations such as Mn2+ and Mg2+, and this spreading was inhibited by synthetic peptides such as RGDS, LRGDV and ELRGDV. These findings suggested that the RGDV region mainly interacted with the cell surface integrins to regulate cell attachment and spreading.

Serum levels of steroid sulfatase protein in gynecologic carcinomas.

Steroid sulfatase (STS) desulfates a number of 3 beta-hydroxysteroid sulfates, converting inactive steroid hormone to the active form. We have established an enzyme-linked immunosorbent assay (ELISA) of STS by using polyclonal antibody against STS purified from human placenta to measure the amount of the enzyme protein in sera. ELISA was performed by a 'Sandwich' method using a peroxidase conjugated anti-STS IgG Fab' fragment. A range of STS of 10-1,500 ng/ml in serum was assayed by this method. When the serum STS from the patients with gynecologic carcinomas was assayed by the ELISA, the level was significantly elevated in endometrial carcinoma (P < 0.05) and ovarian carcinoma (P < 0.01), respectively, as compared with that of normal healthy women.

Hepatocyte growth factor specifically binds to sulfoglycolipids.

Hepatocyte growth factor (HGF) is a heparin-binding pleiotropic factor that acts on a variety of epithelial cells. The interaction of human HGF with glycolipids was studied by overlaying them with 125I-HGF on thin layer chromatograms and by a solid-phase assay using lipids adsorbed on microtiter plates. Among various glycolipids tested, HGF was found to bind to sulfoglycolipids, including galactosylceramide sulfate (SM4), lactosylceramide sulfate (SM3), and gangliotriaosylceramide bis-sulfate. In contrast, HGF failed to bind to gangliosides or neutral glycolipids. HGF binding to SM4 was strongly inhibited by dextran sulfate, heparin, and fucoidan, whereas neither keratan sulfate nor hyaluronic acid had any inhibitory activity. When glycolipids from a renal cancer cell line, SMKT-R3, which overexpresses sulfoglycolipids, were developed on a thin layer chromatogram, SM4 and SM3 were the only glycolipids that bound HGF. We further examined the effect of the incorporation of glycolipids into SMKT-R3 cells on HGF binding to the cells. The incorporation of SM4 into the cells enhanced HGF binding to SMKT-R3 cells, while that of galactosylceramide, a precursor of SM4, had no effect. These observations indicated that SM4 exogenously incorporated into the cell membranes could react with HGF and suggested that endogenous sulfoglycolipids on SMKT-R3 cells might function as reservoirs for HGF.

Cell-surface sulfoglycolipids are involved in the attachment of renal-cancer cells to laminin.

We investigated the role of sulfoglycolipids on human renal-cell carcinoma cells (SMKT-R3) in the attachment to a substrate adhesive protein, laminin. SMKT-R3 cells over-express sulfoglycolipids, including SM2, SM3 and SM4. When acidic glycolipid fractions, were extracted from SMKT-R3 cells, separated by HPTLC, and then overlaid with laminin, laminin bound specifically to SM3 and SM4. A monoclonal antibody, Sulph-1, reacting with SM3 and SM4 inhibited attachment of the cells to laminin but not to fibronectin, in a dose-dependent manner. In addition, when exogenous SM4 was incorporated into the cells, their attachment to laminin, but not to fibronectin, was enhanced. On the other hand, the incorporation of GalCer, which is a precursor of SM4, had no effect on adherence of the cells to laminin or to fibronectin. We also assayed haptotaxis, tumor-cell migration along a gradient of substratum-bound laminin. The incorporation of SM4 into the cells caused an approximately 3-fold increase of the haptotactic response to laminin compared with non- or GalCer-incorporation. These results taken together suggest that sulfoglycolipids on renal-cancer cells are involved in attachment to laminin and that they can modulate the metastatic potential of renal-cell carcinoma cells.

Hepatocyte growth factor elevates the activity levels of glycolipid sulfotransferases in renal cell carcinoma cells.

Accumulation of sulfoglycolipids associated with markedly elevated activity levels of glycolipid sulfotransferases has previously been demonstrated in the human renal cell carcinoma cell line, SMKT-R3. To elucidate the regulatory mechanisms of sulfoglycolipid synthesis in SMKT-R3 cells, the effects of various growth factors on the metabolic enzymes of sulfoglycolipids were investigated. Hepatocyte growth factor (HGF) significantly increased the activity levels of the sulfotransferases in a dose-dependent manner, but did not change that of arylsulfatase A, which hydrolyzes sulfoglycolipids. Scatchard analysis of 125I-HGF binding to SMKT-R3 cells indicated that the cells expressed high-affinity receptors for HGF with a Kd of 36 pM and 750 sites/cell. Furthermore, metabolic labeling with [35S]sulfate revealed that the addition of HGF to the culture medium of the cells resulted in an increment of sulfoglycolipid synthesis. Therefore, these observations suggest that HGF can function as a regulatory factor in sulfoglycolipid synthesis through the modulation of the sulfotransferase activity levels in renal cell carcinoma cells. In addition, HGF stimulated the proliferation and motility of SMKT-R3 cells, suggesting that HGF has multiple biological activities in renal cell carcinoma cells.

Steroid sulfatase deficiency in Japanese patients: characterization of X-linked ichthyosis by using polymerase chain reaction.

X-Linked ichthyosis (XLI) due to deficiency of steroid sulfatase (STS) of which gene consists of 10 exons is an inherited skin disorder. The gene, mRNA and protein of STS were examined in six Japanese patients with XLI. Neither the mRNA nor the enzyme protein was detected in a patient. The results of Southern analysis using STS cDNA as a probe indicated that all the patients examined exhibited large deletions of the STS gene. When exon 1 and the exon 10 of the STS gene were amplified by polymerase chain reaction using patients' genomic DNA as templates, no product was detected in all the patients examined. These observations suggest that most XLI in Japanese patients is caused by an extensive deletion of the STS gene as was demonstrated in Caucasian patients. The PCR method in the present study is useful for the diagnosis of XLI in prenatal and postnatal subjects.

Regulation of activity levels of glycolipid sulfotransferases by transforming growth factor alpha in renal cell carcinoma cells.

Accumulation of sulfolipids associated with markedly elevated levels of glycolipid sulfotransferase activities was previously demonstrated in human renal cell carcinoma cells. To explore the regulation mechanisms of sulfoglycolipid synthesis in renal cancer, effects of various growth factors on the metabolic enzymes of sulfoglycolipids were investigated by using a human renal cell carcinoma cell line, SMKT-R3. Among the growth factors tested, transforming growth factor alpha (TGF-alpha) and epidermal growth factor (EGF) were found to increase the sulfotransferase activity markedly (about 300%), but did not change that of arylsulfatase A, which hydrolyzes sulfoglycolipids. The augmented effects of TGF-alpha was abolished by cycloheximide. Since TGF-alpha is known to bind to the same receptor as EGF, SMKT-R3 cells were investigated for the EGF receptor by affinity cross-linking with 125I-EGF. A radiolabeled protein with a molecular mass of 175 kDa corresponding to the ligand-receptor complex was immunoprecipitated with a monoclonal anti-EGF receptor antibody. When production of the growth factors was examined immunochemically, the cells were found to secrete TGF-alpha at a low level and retain it in a membrane-bound form, whereas EGF was not detected. These observations suggest that the sulfotransferase activities are regulated through the autocrine, paracrine, and/or juxtacrine modes of intercellular stimulation by TGF-alpha in human renal cancer cells.

An adult-type metachromatic leukodystrophy caused by substitution of serine for glycine-122 in arylsulfatase A.

Metachromatic leukodystrophy (MLD) is a lysosomal storage disease with autosomal recessive inheritance caused by a deficiency of the enzyme arylsulfatase A (ASA). We have identified a new mutation in the ASA gene of a patient with adult-type MLD. In this mutation, the glycine at position 122, a highly conserved residue in the AS gene family, was replaced by serine. In a transient expression study, COS cells transfected with the mutant cDNA carrying 122Gly-->Ser did not show an increase of ASA activity and produced little material immunoreactive to an anti-ASA antibody, despite normal mRNA levels.

Expression, purification and binding to the receptor of human insulin-like growth factor II.

Human insulin-like growth factor II (IGF-II) was expressed as a fused protein with 14 additive amino acids in Escherichia coli with a high yield by an expression system using T7 RNA polymerase. Purification of the expressed protein was simply performed using only differential ultrafiltrations, giving a homogeneous preparation upon polyacrylamide gel electrophoresis and high-performance liquid chromatography. The expressed peptide was reacted with a monoclonal antibody raised against native IGF-II on a blotted membrane. Furthermore, the peptide was bound to IGF-II receptor in solubilized rat fetus membrane, though the affinity was slightly inferior to that of native IGF-II. In addition, fusion IGF-II immobilized on a gel matrix was useful for one-step purification of the IGF-II receptor with a high yield from solubilized rat fetus membranes.

Epidermal growth factor elevates the activity levels of glycolipid sulfotransferases in renal-cell-carcinoma cells.

Accumulation of sulfoglycolipids associated with markedly elevated levels of glycolipid-sulfotransferase activity was previously demonstrated in human renal-cell-carcinoma cells. To elucidate the regulatory mechanisms of sulfoglycolipid metabolism in renal-cell carcinoma, effects of various growth factors on the sulfotransferase-activity levels were investigated using a human renal-cell-carcinoma cell line, SMKT-R3. Exogenous epidermal growth factor (EGF) significantly increased the activity levels of the sulfotransferases in a dose-dependent manner, but did not change that of arylsulfatase A, which hydrolyzes sulfoglycolipids. Furthermore, metabolic labeling with 35S-sulfate revealed that the addition of EGF to the culture medium of the cells resulted in an increment of sulfoglycolipid synthesis. The expression of the EGF receptor on SMKT-R3 cells was demonstrated by affinity cross-linking with 125I-EGF. These observations suggest that EGF can regulate sulfotransferase-activity levels in renal-cell-carcinoma cells, and function as one of the regulatory factors of sulfoglycolipid synthesis in these carcinoma cells.

Involvement of protein kinase C in the regulation of glycolipid sulfotransferase activity levels in renal cell carcinoma cells.

Accumulation of sulfolipids associated with elevated levels of glycolipid sulfotransferase activities has previously been demonstrated in renal cell carcinoma cells. To investigate the role of protein kinase C in the synthesis of sulfolipids, the effects of 12-O-tetradecanoylphorbol-13-acetate and protein kinase C inhibitors on glycolipid sulfotransferase activity levels were examined in a human renal cell carcinoma cell line, SMKT-R3. Continuous treatment of the cells with 12-O-tetradecanoylphorbol-13-acetate caused a dose- and time-dependent reduction of the sulfotransferase activity levels. Similarly, protein kinase C inhibitors, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride and staurosporine, reduced the enzyme activities in a dose-dependent manner. These observations suggest that the glycolipid sulfotransferase activity levels are regulated by protein kinase C in SMKT-R3 cells. Furthermore, long-term 12-O-tetradecanoylphorbol-13-acetate treatment resulted in a reduction of sulfolipid synthesis and a decrease of the expression of sulfolipids on the cell surface. Taken together, it is suggested that protein kinase C is involved in the synthesis of sulfolipids through the regulation of the glycolipid sulfotransferase activity levels in renal cell carcinoma cells.

Preparation of a photoaffinity probe for the Man6P/IGF-II receptor.

A photoaffinity probe, 2-(4-phosphopentamanniminophenyl)ethyl-(4-azido) salicylamide, was prepared for photolabelling of the mannose 6-phosphate/insulin-like growth factor II receptor. The probe was synthesized from phosphopentamannan and 2-(4-aminophenyl)ethylamine followed by coupling with photosensitive 4-azidosalicylic acid. The 125I-labelled probe was reacted with receptor purified from rat liver under irradiation with ultraviolet light. Fluorography of the reaction product on a polyacrylamide gel effectively detected the receptor coupled to the photoprobe as a molecular mass of 250 kDa. Specific binding of the probe or of radiolabelled insulin-like growth factor II was not inhibited by the growth factor or by mannose 6-phosphate, confirming different binding sites to the receptor between the ligands.