Search

The main purpose of this in situ hybridization study was to investigate mRNA expression of three bone/cartilage matrix components (perlecan, DMP1, and MEPE) in developing primary (tibial) and secondary (condylar) cartilage. Perlecan mRNA expression was first detected in newly formed chondrocytes in tibial cartilage at E13.0, but this expression decreased in hypertrophic chondrocytes at E14.0. In contrast, at E15.0, perlecan mRNA was first detected in the newly formed chondrocytes of condylar cartilage; these chondrocytes had characteristics of hypertrophic chondrocytes, which confirmed the previous observation that progenitor cells of developing secondary cartilage rapidly differentiate into hypertrophic chondrocytes. DMP1 mRNA was detected in many chondrocytes within the lower hypertrophic cell zone in tibial cartilage at E14.0. In contrast, DMP1 mRNA expression was only transiently detected in a few chondrocytes of condylar cartilage at E15.0. Thus, DMP1 may be less important in the developing condylar cartilage than in the tibial cartilage. Another purpose of this study was to test the hypothesis that MEPE may be a useful marker molecule for cartilage. MEPE mRNA was not detected in any chondrocytes in either tibial or condylar cartilage; however, MEPE immunoreactivity was detected throughout the cartilage matrix. Western immunoblot analysis demonstrated that MEPE antibody recognized two bands, one of 67 kDa and another of 59 kDa, in cartilage-derived samples. Thus MEPE protein may gradually accumulate in the cartilage, even though mRNA expression levels were below the limits of detection of in situ hybridization. Ultimately, we could not designate MEPE as a marker molecule for cartilage, and would modify our original hypothesis.

The Ewing's sarcoma (EWS) oncogene is fused to a variety of cellular transcription factors in various forms of human cancers. Although EWS fusion proteins have been extensively studied, the normal function of EWS remains poorly characterized. We previously reported that a monoclonal antibody, referred to as MY95, recognized nucleoporins such as p62, Nup98, and CAN/Nup214 and an uncharacterized polypeptide with an apparent molecular mass of 83 kDa. In the present study, an amino acid sequence analysis of this 83-kDa protein revealed that it is, in fact, EWS, which is not known to belong to the nucleoporins. We further demonstrated that the immunodeterminant of MY95 contains an N-acetylglucosamine moiety, indicating that EWS is a glycoprotein. Interestingly, the glycosylation level of EWS changes during the neural differentiation of P19 cells. MY95 will be quite useful in further studies of the glycosylated form of EWS in terms of understanding the normal cellular function of this oncogene product.

Stanniocalcin (STC) is a glycoprotein hormone that was first identified in fish, where it regulates the calcium level in the body fluid. The cDNA which encodes human STC has recently been reported but the function has not been completely elucidated. We have prepared a monoclonal antibody against human STC using an analogous peptide of the putative antigenic domain in human STC; it was conjugated with keyhole limpet hemocyanin (KLH). The monoclonal antibody specifically stained the distal convoluted tubules in human kidney which is a putative target organ of STC. The ELISA was established using the monoclonal antibody and recombinant human STC as a standard antigen. The monoclonal antibody prepared in this study provides a useful tool for clinical studies of STC in human.

We have characterized the molecular species and internalization of urinary trypsin inhibitor (UTI) in human uterine fibroblasts. Link protein (LP) has previously been identified as one of the cell-associated UTI binding proteins. The truncated forms of UTI were readily detectable in the cells after incubating the cells with purified UTI. Immunoblotting analysis with a panel of domain-specific antibodies revealed that the UTI species lacked the amino-terminal domain of UTI, but contained the carboxyl-terminal domain. We have examined whether LP is involved in the UTI internalization in the cells. Internalization of 125I-labelled UTI was blocked by the intact UTI, but not by the carboxyl-terminal domain of UTI. Treatment with a polyclonal antibody to the UTI binding domain of LP partially inhibited UTI binding to the cells, but did not significantly prevent UTI internalization. In addition, preincubation of the cells with hyaluronidase reduced the UTI binding to the cells, but had no effect on the rate with which UTI was internalized. These data allow us to conclude that there are at least two different mechanisms for internalization of UTI. The major one is via unknown UTI receptors in a Ca2+, Mg2+-sensitive manner and another is via LP.

Normal rat renal cortex contains three renal disease-inducing substances, that is, nephritogenoside, FX1A and immunogenoside (a novel renal glycoprotein that has the biological activity to induce only active Heymann nephritis). When renal cortex homogenate was treated with a detergent, Triton X-100, nephritogenoside was not eluted in the supernatant in which massive doses of FX1A and immunogenoside were detected. Thus, we searched for nephritogenoside in the precipitate. The precipitate was solubilized with trypsin, and nephritogenoside was easily and effectively isolated through columns of DEAE-cellulose followed by Bio-Gel A-1.5 m. The purified sample thus obtained is only composed of nephritogenoside, and contains neither FX1A nor immunogenoside. The yield of purified nephritogenoside was 3.8 mg (starting from 150 rats), which is about 5-6 times the recovery of the previous methodology. This newly developed simple method may be useful for the isolation of purified nephritogenoside. The chemical and immunologic properties of the purified sample prepared by the new method corresponded well with those of the nephritogenoside which was obtained by the previous methodology after exhaustive digestions with three proteolytic enzymes from homologous renal cortex.

In our experiments, we succeeded in obtaining from human placenta a substance comparable to human renal nephritogenic glycoprotein prepared from GBM (glomerular basement membrane). The procedure was quite similar to that for preparation from the kidney. At first, placental terminal villi were isolated by the successive use of three metal sieves. Then trophoblast basement membranes (TrBM) were isolated from placental terminal villi by ultrasonic disruption. Finally TrBM were made soluble by trypsin digestion, after which they were further digested by pronase. Ouchterlony gel diffusion demonstrated the existence of a common precipitin line between antiserum to human renal nephritogenic glycoprotein prepared from GBM and antiserum to the sample prepared from human placental TrBM, and human renal nephritogenic glycoprotein prepared from GBM and the sample prepared from human placental TrBM. The monosaccharide composition of the sample prepared from human placental TrBM was rich in glucose (glucose, galactose and mannoside in the ratio of 1.00 : 0.74 : 1.00), and the amino acid composition of the sample contained no collagenous components. As a result of fractionation of the sample prepared from human placental TrBM by Bio Gel P200 column chromatography, the activity of renal nephritogenic glycoprotein was found within the void fraction.

Previously, we reported that we succeeded in isolating from human placenta a substance comparable to human renal nephritogenic glycoprotein prepared from glomerular basement membrane (GBM). This time we purified a sample isolated from human placental trophoblast basement membrane (TrBM) with the methods for purifying renal nephritogenic glycoprotein and clarified its chemical composition. To purify the sample prepared from human placental TrBM, we fractionated the sample by Zone Electrophoresis, Concanavalin A(Con A) affinity column chromatography and Bio Gel P200 column chromatography. As the active fraction of the sample purified from human placental TrBM bound specifically with Con A, it proved to be a glycoprotein. The monosaccharide composition of this glycoprotein was rich in glucose (glucose, galactose and mannose were in the ratio of 1.00:0.30:0.33) and the amino acid composition of this glycoprotein contained no collagenous components. Ouchterlony gel diffusion demonstrated the existence of a common precipitin line between antiserum to human renal nephritogenic glycoprotein prepared from GBM and human renal nephritogenic glycoprotein prepared from GBM, purified human placental glycoprotein, the sample prepared from human placental TrBM by sonication and nephritogenic glycoprotein prepared from urine of S.L.E. patient.

Urinary glycoprotein was isolated from human urine by the use of the same procedures as those used for isolation of human renal glycoprotein from the glomerular basement membrane (GBM). In Ouchterlony plates and immunoelectrophoretically, human urinary glycoprotein has a common structural component with human renal glycoprotein isolated from GBM. Massive excretion of the glycoprotein in the urine was associated with only two diseases, systemic lupus erythematosus (membranous type) and membranous glomerulonephritis, although it was isolated from the urine of patients with renal as well as nonrenal diseases and normal subjects. In these two membranous diseases, the glycoprotein of GBM could be intensively liberated into the urinary space, as the result of extensive damage of GBM that is initiated by immune-complex deposit. It is discussed that urinary content of this glycoprotein may indicate the degree of the GBM damage in these diseases.

A glycopeptide comparable to human renal nephritogenic glycopeptide (nephritogenoside) of glomerular basement membrane (GBM) origin was isolated and purified from human placenta. Trophoblast basement membranes (TrBM) were isolated from placental terminal villi by ultrasonic disruption, and digested with trypsin and then with pronase. TrBM were then fractionated by zone electrophoresis, concanavalin A (Con A) affinity column chromatography and Bio-Gel P200 column chromatography. From 250 g (wet weight) of human placental terminal villi, we obtained a purified sample of 0.7 mg (dry weight) of glycopeptide. Ouchterlony gel diffusion demonstrated the existence of a common precipitin line between purified human placental glycopeptide and antiserum against human renal nephritogenic glycopeptide prepared from GBM. The sugar composition of this glycopeptide was rich in glucose (glucose, galactose and mannose were in the ratio of 1.00:0.30:0.33) and the amino acid composition of this glycopeptide contained no collagenous components. These results indicate that at least one of the nephritogenic substances common to the placenta and kidney is a glycoprotein rich in glucose and different from the collagenous component of the basement membrane.

Nephritogenoside is a glycopeptide with the ability to induce chronic progressive glomerulonephritis (end stage kidney) in homologous animals, by a single footpad injection. This substance contains a novel carbohydrate-peptide linkage, i.e. the trisaccharide (3 glucose residues) chain is alpha-N-glycosidically linked to the polypeptide chain through the amido nitrogen of an asparagine residue at the N-terminal. It was found that the peptide portion of nephritogenoside is composed of twenty-one amino acids (Asn1-Pro-Leu-Phe-Gly5-Ile-Ala-Gly-Glu-Asp10-Gly-Pro-Thr-Gly-Pr o15-Ser-Gly-Ile- Val-Gly20-Gln21) and that the peptide portion has a repeated Gly-X-Y structure.

A single footpad injection of a triglycosyl glycopeptide (nephritogenoside, NG) isolated from rat glomerular basement membrane can induce proliferative glomerulonephritis (PGN) in homologous animals, resulting in contracted kidney. On the other hand, it is well known that administration of rat tubular brush border antigen (Fx1A) causes a membranous glomerulonephritis (MG) in homologous animals. When a partially purified NG (crude NG, cNG) is injected in rats, a mixed lesion of PGN and MG resulted. To investigate the correlation between renal glomerular lesions and humoral immune responses in animals given NG and cNG, levels of antibodies to cNG and Fx1A, and those of immune complexes (ICs) are measured in sera from animals of these experimental groups using enzyme-linked immunosorbent assay. Significant levels of antibodies to cNG and Fx1A and those of ICs are detected in all groups of rats injected with cNG, Fx1A and pronase-digested Fx1A. It was revealed that antibodies to cNG in sera from rats injected with cNG, Fx1A and pronase-digested Fx1A recognize the common epitopes shared with Fx1A. On the other hand, neither antibodies nor ICs are detected in rats injected with NG, suggesting that humoral immune mechanisms may not play a leading role in the pathogenesis of NG-induced glomerulonephritis.

Nephritogenic glycopeptide, nephritogenoside, is a minor component of basement membranes (the yield: 2-3 micrograms/kidney), and in homologous animals causes chronic glomerulonephritis. Nephritogenoside was compared to other basement membrane components including collagenous and noncollagenous glycoproteins. In limited pepsin digests of rat renal cortices, nephritogenoside was separable from type IV collagen by DEAE-cellulose column chromatography, although these two substances showed immunological cross-reactivity each other. Nephritogenoside did not immunologically cross-react with laminin and fibronectin by direct or inhibition enzyme immunoassay. These results indicate that nephritogenoside is different from basement membrane glycoproteins such as laminin and fibronectin and that nephritogenoside can be isolated from type IV collagen.

Cachectin/tumor necrosis factor (cachectin/TNF) has been shown to be capable of stimulating prostacyclin (PGI2) production by vascular endothelial cells in vitro. The stimulation of PGI2 by cachectin/TNF is comparable to that observed with interleukin-1, the monokine previously suggested to be the principal mediator of this effect. The ability of cachectin/TNF to stimulate PGI2 production suggests that it may play a role in producing depressed blood pressure or shock. If so, it might be possible to prevent such adverse effects with the aid of anti-inflammatory agents.

A new immunochemical procedure was introduced to estimate the nephritogenic activity of collagenase and pronase digests of various rat organs. A glycoprotein isolated from collagenase digests of various rat organs showed the nephritogenic activity as well as the antigenic activity that induces nephrotoxic antibody, which were nearly identical to those of a glycoprotein isolated from trypsin digests of the rat organ concerned. A glycoprotein isolated from pronase digests of various rat organs was proved to have no antigenic activity that induces nephrotoxic antibody, but the existence of nephritogenic activity was proved in this glycoprotein. These results were supported firmly by the assay experiments for nephritogenicity. Ouchterlony gel diffusion test and the assay experiments demonstrated the existence of a common nephritogenic substance among the glycoproteins isolated from trypsin, collagenase and pronase digests of rat organs.

A new glycopeptide was isolated from the glomerular basement membrane (GBM) of normal rats. Unlike already known glycopeptides, this glycopeptide has biological activity (nephritogenic activity) to induce glomerulonephritis when injected once into the footpads of homologous animals. A close relationship was found between the nephritogenic activity and the non-dialyzable glucose content of this glycopeptide. Thus the nephritogenic activity can be assessed quantitatively by estimating the content of "non-dialyzable glucose." Chemical purification of the nephritogenic glycopeptide involved the selective removal of inactive glycopeptide containing galactose, mannose, and N-acetyl-glucosamine (but no glucose). Trichloroacetic acid (TCA) treatment was a simple but highly effective procedure for selective removal of this inactive glycopeptide. The non-reducing terminus of the nephritogenic glycopeptide is alpha-D-glucopyranoside, and the glycopeptide reacts specifically with concanavalin A, even in the crude state. We propose that the nephritogenic glycopeptide is not an artifact produced during exhaustive proteolytic digestion, but a natural substance having a fixed molecular shape, even in the crude state, and whose union with GBM-proper can be easily broken by proteolytic digestion.

Rat glomerular basement membrane was extracted for 3 h with trypsin, pH 8.0. The supernatant solution was treated with trichloroacetic acid and the supernatant thus obtained was applied to Bio-Gel P200. The first of the two glycoprotein peaks was applied onto Sepharose derivatives of concanavalin A (Con A). Examination of the material retained by the unsolubilized Con A and subsequently eluted with methyl alpha-D-mannopyranoside reveals that the principal high affinity receptor for Con A is the renal glycoprotein, having antigenic activity that induces nephrotoxic antibody. This glycoprotein has also nephritogenicity (the activity capable of inducing glomerulonephritis in homologous animals by a single foot pad injection with Freund's incomplete adjuvant). Evidence is given to show that this binding is specific. The remainder of the renal glycoprotein is unretarded and is revealed to contain none of the activities described above. When fluorescein isothiocyanate-labelled Con A is, conversely, injected into rats through the renal artery, the specific binding of Con A to the glomerular capillary loop is proved. The results demonstrated appear to, indicate that the receptor for Con A present in normal rat glomerular basement membrane can be identified as the well-established chemical substance, the nephritogenoside, having the alpha-D-glucopyranosyl unit at the non-reducing terminus which is facing the endothelial aspects of the glomerular capillary loop.

Studies on the immunochemical characterization were made on the antigenic substance of membranous glomerulonephritis (MG) that is induced, in homologous animals, by a single footpad injection of rat crude nephritogenoside (CNG). In rats injected with rat CNG, typical MG with immunofluorescent granular pattern (immune complex type nephritis) was produced 3-4 months after the injection. Since CNG nephritis is a model which induces MG only in homologous animals, the development of MG in this model should be explained by antigen-autoantibody immune complex mechanism. Thus, the features of the antigenic substance contained in CNG was immunochemically investigated, utilizing the serum (autoantibody) of rats with MG as well as rabbit antiserum to rat tubular brush border antigen (FXIA) (heteroantibody). As a result, two different fractions (fr. 31 and fr. 36) were separated from the antigenic substance contained in CNG: that is, fr. 31 reacts only with rabbit antiserum to rat tubular brush border antigen (FXIA) (heteroantibody) but does not react with the serum (autoantibody) of rats with MG. Fr. 36 does not react with rabbit antiserum to FXIA (heteroantibody) and reacts only with the serum (autoantibody) of rats with MG (which was induced by a single footpad injection of CNG or soluble FXIA). These results suggest immunologically that fr. 36 is the main antigen responsible for the development of MG which is induced in homologous animals by a single footpad injection of CNG.

An enzyme linked immunosorbent assay (ELISA) was introduced for the detection of nephritogenic glycopeptide, nephritogenoside and Heymann's antigen. In the fraction of crude nephritogenoside which induced membranous glomerulonephritis (besides proliferative glomerulonephritis) in rats, both nephritogenoside and Heymann's antigen were detected by ELISA. On the other hand, in the sample of pure nephritogenoside which induced only proliferative glomerulonephritis, Heymann's antigen was not detected at all. These results indicate that crude nephritogenoside preparation contains Heymann's antigen as an inducing factor of membranous glomerulonephritis in homologous animals. In addition to TCA treatment or DEAE-cellulose column chromatography, gel filtration on Bio-Gel P200 was a good tool for the removal of Heymann's antigen from nephritogenoside.

Glomerular basement membrane (GBM) damage has been considered as the only source of the nephritogenic glycoprotein excreted in urine. However, in the course of our investigation on the glycoprotein or glycopeptide isolated from the urine of a disease group seldom affected with GBM damage, it appeared that a second source of the nephritogenic glycoprotein excreted in the urine is in blood plasma; that is, in Zn-alpha2-glycoprotein. Zn-alpha2-glycoprotein is the only plasma protein which has a common determinant with nephritogenic urinary glycoprotein or glycopeptides. It was proposed that plasma Zn-alpha2-glycoprotein may play the role of a carrier protein of the nephritogenic renal glycoprotein.

Proliferative glomerulonephritis was induced in rats by a single injection in the hind footpads of the glycoprotein isolated from pronase digests of homologous renal cortex, in spite of the evidence that the glycoprotein no longer possessed the antigenic activity with respect to producing the nephrotoxic antibody. In rats given the glycoprotein (without trichloroacetic acid (TCA) treatment) prepared from pronase digests of homologous renal cortex, morphologic changes of proliferative glomerulonephritis were followed (3--4 months after injection) by those of typical membranous glomerulonephritis with an immunofluorescent 'granular' pattern, but in animals which received the glycoprotein (with TCA treatment), the morphologic changes of the kidney were those of proliferative glomerulonephritis with an immunofluorescent 'mesangial' pattern throughout the whole course. Morphologic changes of membranous glomerulonephritis induced in the group given the glycoprotein (without TCA treatment) can be explained by an immune-complex mechanism, superimposed on the basic pathogenesis (common to both groups) of proliferative glomerulonephritis with immunofluorescent 'mesangial' pattern.

When triglycosyl glycopeptide ( nephritogenoside ) was contaminated with glucosyl, galactosyl, mannosyl, and heteropolysaccharide glycopeptide, a mixed lesion of proliferative glomerulonephritis ( PGN ) with immunofluorescent mesangial pattern and membranous glomerulonephritis (MG) with immunofluorescent granular pattern resulted. It was concluded that PGN was caused by the presence of nephritogenoside and MG by the presence of the heteropolysaccharide glycopeptide in the crude nephritogenic glycopeptide mixtures derived from various solubilization steps used to treat the kidney extracts. Trichloroacetic acid treatment of crude nephritogenic glycopeptide mixtures precipitated the heteropolysaccharide glycopeptide of the MG-inducing factor, thus permitting the separation of nephritogenoside from the heteropolysaccharide glycopeptide by a rather simple technique. Both the heteropolysaccharide glycopeptide and Heymann's antigen (so-called tubular antigen) have a common antigenic substance, but we cannot conclude, at the present state, that both substances are chemically the same. The degree of contamination of pure nephritogenoside by this substance that is antigenically the same as Heymann's antigen (in pure nephritogenoside ) can be chemically and accurately determined, based on the evidence that pure nephritogenoside is a glycopeptide having only three glucose residues as the sugar moiety, that is, by estimating the amount of contaminated monosaccharide components such as galactose, mannose, and N-acetylglucosamine.

Proliferative glomerulonephritis was induced in rats by a single injection in the hind footpads of the urinary glycoprotein or more purified urinary glycopeptide with Freund's incomplete adjuvant. Although the urinary (water-soluble and non-dialyzable) glycoprotein and glycopeptide were prepared from heterologous urines (human urines), the immunofluorescent pattern of the kidneys was not 'granular' but 'mesangial', as in rats given a single injection of homologous or heterologous renal glycoprotein with nephritogenicity. The pathogenesis of proliferative glomerulonephritis in the present experiments may be explained by the same mechanism as that of rats given a single injection of renal glycoprotein, i.e. by any mechanism other than antigen (heterologous protein)-antibody immune complex and anti-GBM antibody mechanisms.

It is postulated that an initial step in dental plaque formation is the adherence of oral bacteria to the salivary pellicle. Recently, we have found that a proline-rich and basic glycoprotein (MGP) from human parotid saliva, which is successfully purified by Concanavalin A-Sepharose affinity chromatography, binds to some oral streptococci such as S. mitis and S. sanguis. This paper deals with some inhibitors which affect the binding of the MGP to S. sanguis ATCC 10557. The assay for the binding ability of the radioactive MGP to the bacterial cells was performed by incubation of the reaction mixture containing 10 microgram of [3H]MGP (6000 dpm) and about 4.5 mg of bacterial cells (dry weight) in 0.5 ml of 0.05 M Tris-HCl buffer containing 0.05 M NaCl, pH 8.0 with a final volume of 0.51 ml. After 1 hour standing at 4 degrees C, the cells were washed five times with the same buffer. The resulting sediment was solubilized in 0.5 ml of NCS tissue solubilizer and the radioactivity was measured. The binding of the radioactive MGP to bacterial cells was specifically inhibited by galactose, lactose and N-acetyllactosamine. Applications of heat and trypsin on the cell surface, strikingly reduced the binding ability. These findings strongly suggested that a lectin-like substance may be present on the bacterial cell surface.

Water-soluble and non-dialyzable glycopeptide, nephritogenoside, was isolated from the glomerular basement membrane of normal rats. The yield of the purified nephritogenic glycopeptide from the glomerular basement membrane of 1200 rats was only 17.2 mg. Hexose amounted to 24.3% by weight, and consisted only of glucose. Paper chromatographic studies on the number and length of the carbohydrate chain deduced from strong alkaline cleavage in the presence of sodium borohydride strongly suggested that the carbohydrate chain of the nephritogenic glycopeptide is composed of three glucose residues. This conclusion was supported by the 13C-NMR spectroscopic results. In the paper chromatographic studies on the monosaccharides produced from 3H-labeled oligosaccharide by alkaline degradation and then acid hydrolysis and studies on the 13C-NMR spectrum, it was demonstrated that the saccharide at the reducing terminus is glucose. Thus, the glucose residue at the reducing terminus of the nephritogenoside may be linked directly (probably N-glycosidically) to amino acid, without the intervention of N-acetylglucosamine. The proposed structure of the carbohydrate portion of the nephritogenic glycopeptide, nephritogenoside, is as follows: (formula in text)

O-(alpha-D-Glucopyranosyl)-(1----6)-O-beta-D-glucopyranosyl-(1----6)-1-N -(glycylglycylglycyl-L-seryl-L-leucyl-L-glutam-5-oyl)-alpha- D -glucopyranosylamine has been prepared, as a model of a derivative possibly present in the glomerular basement membrane of rats, by condensation of the corresponding L-glutamyl derivative of the trisaccharide with the pentapeptide in the presence of O,O-diethyl cyanophosphonate. Protective groups were removed by O-deacetylation, deethoxylation, and hydrogenolysis. The glutamyl derivative of the trisaccharide was also converted into the acyl azide which was condensed with bovine serum albumin to form a neoglycoprotein.

Nephritogenoside is a minor component of the basement membrane of normal animals (including humans). It is a glycopeptide with the ability to induce chronic progressive glomerulonephritis (end stage kidney) when administered as a single footpad injection, and contains a novel carbohydrate-peptide linkage. The total chemical structure was investigated. It was revealed that nephritogenoside is a simple glycopeptide composed of three glucose residues [alpha-Glc-(1----6)-beta-Glc-(1----6)-Glc] and twenty-one amino acids [1Asn-Pro-Leu-Phe-5Gly-Ile-Ala-Gly-Glu-10Asp-Gly-Pro -Thr-Gly-15Pro-Ser-Gly-Ile-Val-20Gly-21Gln], and that the glucose residues are linked alpha-N-glycosidically to the N-terminal amino acid.

High-metastatic murine fibrosarcoma cells readily attached to Type IV (basement membrane) collagen, whereas low-metastatic cells isolated from the same tumor did not. The addition of laminin--a glycoprotein that facilitates the adherence of epithelial cells to their basement membranes--enhanced the attachment of the low-metastatic cells, but not the high-metastatic cells. Using anti-laminin antibodies and a laminin-binding lectin as probes, the authors were able to identify by immunofluorescence a moiety associated with the high-metastatic cells, but not the low-metastatic cells, which cross-reacted with murine laminin purified from the EHS sarcoma. When extracts from the high-metastatic cells were separated by affinity chromatography, with the laminin-binding lectin as the affinity substrate, a substance was isolated that had an apparent molecular weight of 56,000 daltons. The affinity-purified material reacted strongly with anti-laminin antibodies by enzyme-linked immunosorbent assay.

We recently reported that an injection of antibody raised against renal brush border glycoprotein, gp108, in rats induced passive Heymann nephritis with acute and severe proteinuria. In this study, the distribution of gp108 in various rat tissues was investigated. On enzyme immunoassay, anti-gp108 antibody reacted to extracts of small intestine, lung, spleen, thymus, liver, epididymis, stomach, pancreas and heart. It also reacted to sera and extracts of peripheral blood cells, especially lymphocytes. These antigens, which are cross-reactive to kidney gp108 have similar biochemical properties: a molecular weight of about 108,000 and an isoelectric point of 4.8-5.4. These results show that immunologically and biochemically related antigens to renal glycoprotein, gp108, are present in various rat tissues.

Tumor necrosis factor (TNF) has been reported to be identical to "cachectin," a monokine which we have previously proposed as a mediator of the enhanced catabolism observed in patients or animals responding to various invasive stimuli such as infections. Detailed quantitative studies were conducted on the effects of TNF on fatty acid metabolism in 3T3-L1 cells in order to explore the extent of the catabolic effects exerted by TNF compared with those by the crude cachectin. 3T3-L1 adipocytes responded to recombinant human TNF, showing a decrease in LPL activity and an increase in intracellular lipolysis. When TNF in the crude cachectin preparation was completely neutralized with anti-TNF antibody, about 75% of LPL suppression activity in the crude cachectin was absorbed, indicating that most of the mediator responsible for LPL suppression in the crude preparation is TNF. In contrast to the above effect on LPL, TNF markedly increased the lipolysis of stored fat in the cells. The effect on LPL was observed as early as 2 h after the addition of TNF, but enhancement of lipolysis required a time lag of at least 3 h before any increase of glycerol release became apparent. The effective concentrations of TNF for the stimulation of lipolysis were much higher (2.5 to 49 nM) than those for LPL suppression (50 pM to 50 nM), but both were in the same range as the concentration required for tumoricidal effect. These results demonstrate that cachectin is synonymous with TNF and that it plays an important role in the pathophysiology of deranged lipid metabolism through both suppression of LPL and enhancement of lipolysis in patients coping with invasive conditions such as infections.

In rats given a single injection (500 μg) in the hind footpads of heterologous (water-soluble) renal glycoprotein and Freund's incomplete adjuvant, proliferative glomerulonephritis with immunofluorescent `mesangial pattern' was induced. Immunopathological and morphological changes of this group were very similar to those of rats which received a single injection of homologous and water-soluble renal glycoprotein. The nephritogenic renal glycoprotein does not show species specificity. [ABSTRACT FROM AUTHOR]

Rats given a single injection in the hind footpads of homologous, water-soluble, organ glycoprotein and Freund's incomplete adjuvant developed proliferative glomerulonephritis. Among the glycoproteins obtained from various rat organs, the nephritogenic activity (on a dry weight basis) of the active glycoprotein prepared from lung was a little lower than that from renal cortex, and was followed by those from aorta, heart, liver, spleen and muscle in that order. This nephritogenic glycoprotein is present also in renal medulla, and also in renal pyramids (inner medulla) which contain neither glomeruli nor proximal convoluted tubules. The activity of nephritogenic glycoproteins prepared from various rat organs is nearly parallel with both the hexose content and the yield of the cathodal part of the hexose peak in zone electrophoresis. Nephritogenic glycoproteins obtained from various rat organs share a structural component isolated from GBM. However, the immunofluorescence pattern in the glomerulonephritis induced by a single injection of each of these glycoproteins was 'mesangial' rather than 'linear'. [ABSTRACT FROM AUTHOR]