Engineered IGF-I expression induces glandular enlargement in the murine prostate.

IGF-I has been implicated as a factor that may predispose one to prostate cancer and to benign prostatic hypertrophy (BPH). We established murine IGF-I transgenic mice under the control of rat probasin promoter and analysed the histology of the murine IGF-I-overexpressing prostate. Immunohistochemically, IGF-I was expressed in prostatic epithelial cells or basement membranes of the ventral, dorsal and lateral lobes in a line of IGF-I transgenic mice, but not in their control littermates. The anterior lobe did not express IGF-I. IGF-binding protein-3 (IGFBP-3), inhibitory to the mitogenic action of IGF-I, was detected in epithelial cells of prostatic ventral lobes, but not in those of the dorsal, lateral or anterior lobes of IGF-I transgenic mice. In controls, IGFBP-3 was not detected in epithelial cells of any prostatic lobe. Macroscopic prostatic size and the appearance of IGF-I transgenic mice were comparable with those of their control littermates of the same age. With a computed morphometric analysis, epithelial glands and intraglandular lumens in the prostatic lobes except the ventral lobe were smaller at 17 Months of age than at 14 Months both in IGF-I transgenic mice and controls. Glands and intraglandular lumens in the ventral prostatic lobes of IGF-I transgenic mice expressing more IGF-I protein in the prostate than controls were dense and enlarged similar to cysts compared with those of non-transgenic littermates without showing epithelial growth. Glands and lumens in the dorsal and lateral lobes of the IGF-I transgenic mice were also larger than controls at 14 and/or 17 Months of age. Glands in the anterior prostatic lobe of the IGF-I transgenic mice were not morphologically or morphometrically different from those of non-transgenic littermates. In conclusion, IGF-I transgenic mice under the control of rat probasin promoter showed more dense and enlarged epithelial glands in their prostatic ventral, dorsal and lateral lobes.

[Parathyroid cyst].

Parathyroid cysts are grouped into two: functioning and non-functioning cysts. Most of the functioning cysts result from the cystic degeneration of parathyroid adenoma, while, non-functioning cysts, in many cases, originate from Kürsteiner's canal on the third branchial cleft. It is difficult to differentiate parathyroid from thyroid cysts morphologically. However, the measurement of assay of parathyroid and thyroid hormone in the aspirated fluid enables differentiation. Hypercalcemic crisis occurs frequently in the functioning cysts. Moreover, we propose sclerotherapy as the treatment for the non-functioning cysts.

N-acetylneuraminic acid and N-glycolylneuraminic acid in glycopeptides of colonic tumor and mucosa in rats treated with carrageenan and 1,2-dimethylhydrazine.

N-linked glycopeptides were prepared from colonic tumor (adenocarcinoma) and mucosa in rats treated with carrageenan, an indigestible polysaccharide, and 1,2-dimethylhydrazine. Sialic acids, N-acetylneuraminic acid and N-glycolylneuraminic acid, obtained by acid hydrolysis of the glycopeptides were determined by HPLC. The N-acetylneuraminic acid/N-glycolylneuraminic acid ratio in colonic tumor was 25.2, while each treated mucosa had the values between 0.29 and 0.55. Thus, necessity which observes the qualitative change of sialic acid in malignant transformation was suggested.

Promoter function of carrageenan on development of colonic tumors induced by 1,2-dimethylhydrazine in rats.

In order to understand the function of carrageenan, an indigestible polysaccharide, as a promoter of colonic tumors induced by 1,2-dimethylhydrazine (DMH), molecular weight distribution of fecal carrageenan and amounts of fecal bile acids in rats given carrageenan and DMH treatment were examined. Gel filtration pattern on Sephacryl S-300 of fecal carrageenan was very similar to that of feeding carrageenan, and carrageenan ingested was quantitatively excreted in feces. Hexafluoroisopropyl ester-trifluoroacetyl derivatives of fecal bile acids were analyzed by gas chromatography on QF-1. Although there was a decreased concentration of deoxycholic acid and total bile acids in carrageenan-fed rats compared to control rats, no difference in the daily output was found because carrageenan ingestion increases fecal output. Significant increased concentration and daily output of lithocholic acid, a tumor-promoter, by feeding carrageenan were found. Thus, it was suggested that the promoting effect of carrageenan on colon tumorigenesis by DMH may be mediated by increased excretion of lithocholic acid and may not participate in degradation of carrageenan ingested.

Structural analyses of asparagine-linked oligosaccharides of porcine pancreatic kallikrein.

The structures of asparagine-linked oligosaccharides of porcine pancreatic beta-kallikrein are reported. Asparagine-linked neutral oligosaccharides were released by N-oligosaccharide glycopeptidase digestion, and the reducing ends of the oligosaccharides were derivatized with a fluorescent reagent, 2-aminopyridine. The mixture of pyridylamino oligosaccharides was separated by reverse-phase and amide-adsorption high-performance liquid chromatography. The pyridylamino oligosaccharides were separated into more than 50 kinds of oligosaccharides. The structures of 5 kinds of triantennary and 12 kinds of tetraantennary oligosaccharides were determined by the use of high-resolution proton nuclear magnetic resonance spectroscopy and methylation analysis. Furthermore, the structures of five kinds of oligomannose-type oligosaccharides were elucidated by a combination of exoglycosidase digestion and high-performance liquid chromatography. 1H NMR data for 14 out of the 17 kinds of N-acetyllactosamine-type oligosaccharides reported here have not previously been described in the literature. (1) It has been shown that fucose containing tri- and tetraantennary oligosaccharides is predominant in porcine pancreatic beta-kallikrein B. (2) It has also been shown that the heterogeneity of the structure in these types of oligosaccharides is derived from the variety of the positions of galactose residues linked to outer N-acetylglucosamine residues. (3) The distribution of oligosaccharides into two glycosylation sites, asparagine-95 and asparagine-239, of beta-kallikrein B was determined. It has been found that oligomannose-type oligosaccharides are exclusively present at asparagine-239, although N-acetyllactosamine-type oligosaccharides occur at both glycosylation sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Structural analysis of N-linked oligosaccharides by a combination of glycopeptidase, exoglycosidases, and high-performance liquid chromatography.

A simple, sensitive, and rapid method for the analysis of structures of N-linked carbohydrates is reported. The method involves four steps: preparation of carbohydrate chains from glycopeptides by N-oligosaccharide glycopeptidase digestion; derivatization of the reducing ends of carbohydrate chains with a fluorescent reagent, 2-aminopyridine, by using sodium cyanoborohydride; separation of oligosaccharide derivatives by reverse-phase high-performance liquid chromatography; and structural analysis of oligosaccharides by sequential exoglycosidase digestion. The elution positions of 50 standard oligosaccharide derivatives were determined by HPLC. The structure of an unknown oligosaccharide can be characterized by comparison of its elution position with those of the standard compounds. The method was applied to elucidate the structures of oligosaccharides in the myeloma IgG protein, Yot.

Comparative structural study of the N-linked oligosaccharides of human normal and pathological immunoglobulin G.

The structures of oligosaccharides of normal and pathological immunoglobulin G (IgG) are reported. Asparagine-linked neutral oligosaccharides were released by N-oligosaccharide glycopeptidase (almond) digestion. The reducing ends of the oligosaccharide chains thus obtained were aminated with a fluorescent reagent, 2-aminopyridine, and the mixture of pyridylamino derivatives of the oligosaccharides was separated by reverse-phase high-performance liquid chromatography. It was possible to separate 15 out of the 16 kinds of oligosaccharides that have been suggested to exist in normal human IgG. High-resolution proton nuclear magnetic resonance spectroscopy was used along with chemical methods to determine the structures of the separated oligosaccharides. It has been shown that in normal IgG a biantennary complex-type oligosaccharide with a fucose residue (formula; see text) is predominant and four kinds of oligosaccharides, which are biantennary with bisecting N-acetylglucosamine and without fucose residues, exist only in a very small quantity. The results obtained for normal IgG were compared with those obtained for three myeloma IgG proteins. It has been found that the most abundant species that exist in the pathological proteins analyzed in the present work lack one or two galactose residues at the nonreducing terminal. We show that the fractions of fucose-containing oligosaccharides are markedly decreased in the heavy-chain disease protein Per. It is of particular interest that in this paraprotein the major component is a biantennary complex-type oligosaccharide that lacks a fucose residue and an oligosaccharide with the structure (Formula: see text) exists as one of the most abundant components.

Enhancing effect of carrageenan on the induction of rat colonic tumors by 1,2-dimethylhydrazine and its relation to beta-glucuronidase activities in feces and other tissues.

Since it has been demonstrated that a high level of fat is a dietary factor in the etiology of colon cancer, the effect of carrageenan, a polysaccharide extracted from the red seaweeds, on 1,2-dimethylhydrazine-induced colonic tumors in rats fed a semipurified control diet containing an ordinary level of fat was studied. Nevertheless, the enhancing effect of carrageenan on colonic tumors was observed. The rats fed a carrageenan diet had approximately twice the fecal weight compared to the rats fed a control diet. While no significant differences were found in beta-glucuronidase activities in colonic mucosa, liver or plasma in the carrageenan-fed rats and controls, the activity in feces was significantly lower in the carrageenan-fed rats. At least, no beta-glucuronidase activity seemed to be related to the tumor-enhancing effect of carrageenan.

The core molecule from type H proteoglycan. Release of mannose-containing oligosaccharides by digestion with N-oligosaccharide glycopeptidase.

Chick-embryo cartilage contains a unique set of proteoglycans. Type H proteoglycan (PG-H) is the most abundant, constituting over 90% of the total cartilage hexuronate. We previously showed that treatment of PG-H with chondroitinase ACII and keratanase yields a protein-enriched core molecule [PG(-CS,KS)] with enzymically modified linkage oligosaccharides of the chondroitin sulphate and keratan sulphate chains. We report here that further treatment of PG(-CS,KS) with pepsin and N-oligosaccharide glycopeptidase (almond glycopeptidase) released four distinct types of mannose-containing oligosaccharide. Two of them were shown to be: (Formula: see text). Of the mannose-containing glycopeptides formed by pepsin digestion, about 40% (as mannose) were resistant to N-oligosaccharide glycopeptidase. Since the resistant fraction was enriched in keratan sulphate remnants, it is suggest that the mannose-containing oligosaccharides in this fraction represent those located in a keratan sulphate-enriched region of PG-H.

Comparative study of the oligosaccharides of human thyroglobulins obtained from normal subjects and patients with various diseases.

Asparagine-linked oligosaccharides were released quantitatively by N-oligosaccharide glycopeptidase (almond) digestion from human thyroglobulins prepared from thyroid glands of normal subjects and patients with several pathological conditions. The pyridylamino derivatives of the oligosaccharides were prepared and analyzed by high-performance liquid chromatography. The content of high-mannose-type oligosaccharides was comparable to that of the complex type in normal thyroglobulins. Man9GlcNAc2 was the predominant component in the high-mannose-type region, while biantennary oligosaccharides with fucose were the major components in the complex-type region. High-mannose-type oligosaccharides were markedly decreased in thyroglobulins prepared from patients with various disorders, such as Basedow's disease, papillary carcinoma, and adenomatous goiter, whereas they were appreciably increased in thyroglobulin from diffuse goiter.

1H-NMR spectroscopy of the glycoprotein-bound large carbohydrates from embryonal carcinoma cells.

400 MHz NMR spectrum was recorded for the glycoprotein -bound large carbohydrates (embryoglycan) isolated from F9 embryonal carcinoma cells. Two intense signals at 4.13 ppm and 4.69 ppm were assigned to be H-4 of galactosyl residues substituted at C-3 and H-1 of G1cNAc beta 1----3, respectively. The result is consistent with the proposal that the fundamental building unit of the large glycan is G1cNAc beta 1----3Ga1 beta. Furthermore, the spectral data confirmed a conclusion obtained by glycosidase digestion that fucosyl residues are linked mostly to N-acetylglucosamine rather than galactose.

Friend erythroleukaemia cell mutants defective in viral gene expression.

Cellular mutants defective in the expression of viral polypeptides were isolated from the Friend erythroleukemia cell line 745a by immunoselection with anti-ecotropic murine leukaemia virus serum in the presence of complement. The mode of appearance of the antiserum-resistant cells showed an interesting pattern which is discussed in the text. One of the mutants obtained contained no detectable gp70 or p15(E) while retaining gPr90env; defects appeared to reside in the processing of the gPr90env to gp70 and p15(E). In another type of mutant, gPr90env was not detected. All these mutants retained spleen focus-forming virus (SFFV)-specific gp55 and gag precursor Pr68gag. The mutants superinfected with the helper virus produced the helper and SFFV also, indicating that these mutations did not affect the replication of the exogenously infecting virus.

Glycoprotein-bound large carbohydrates of early embryonic cells: structural characteristic of the glycan isolated from F9 embryonal carcinoma cells.

The high-molecular-weight glycopeptides characteristic of early embryonic cells were isolated from F9 embryonal carcinoma cells grown in vitro and also from the cells grown in vivo as subcutaneous tumors. The two preparations had similar carbohydrate compositions. The major components were galactose and N-acetylglucosamine (molar ratio 1:0.86) in the glycan isolated from the cultured cells. In addition, small amounts of fucose, N-acetylgalactosamine and mannose were present. The glycan from the in vitro grown cells was found to have a molecular weight of more than 10,000 by gel filtration after mild alkaline treatment or hydrazinolysis. The structural characteristics of the core portion of the glycan were studied by using the radioactively labeled glycopeptide from the in vitro grown cells. Methylation analysis provided the following informations. 1) The glycan was highly branched at galactosyl residues. 2) Large numbers of galactosyl residues were also present at non-reducing termini. 3) Monosubstitution of galactose occurred at C-3. 4) Glucosamine residues were mainly monosubstituted. That the disaccharide GlcNAc-Gal was the major structural unit of the glycan was suggested by the isolation of the deacetylated disaccharide after alkaline thiophenol cleavage followed by acid hydrolysis. Furthermore, methylation analysis of the glycan from the in vivo grown tumors indicated that monosubstitution of glucosamine occurred at C-4 and that disubstitution of galactose occurred at least mainly at C-3 and C-6. We propose that the basic structural unit of the core portion is 4GlcNAc 1 leads to 3Gal, and that the galactosyl residue serves as a branching point at C-6. Thus, the structural unit of the core portion of the large glycan appears to be the same as that of lactosaminoglycans found in adult cells.

Demonstration of a new glycopeptidase, from jack-bean meal, acting on aspartylglucosylamine linkages.

An enzyme preparation from jack-bean meal hydrolyzed beta-aspartylglucosylamine linkages in glycopeptides. The enzyme could release sialic acid-containing complex-type oligosaccharides as well as high-mannose-type and hybrid-type oligosaccharides. The products were equimolar amounts of ammonia, oligosaccharide and peptide. The enzyme cleaved glycopeptides with three or more amino acid residues, whereas it did not hydrolyze GlcNAc-Asn. The mechanism of action of the enzyme and substrate specificity so far tested were similar to those of the glycopeptidase from almonds.

Structure and location of asparagine-linked oligosaccharides in the Fc region of A human immunoglobulin D.

Seven kinds of asparagine-linked oligosaccharides were bound to the Fc region of a human immunoglobulin D(NIG-65). The oligosaccharides quantitatively released from four species of glycopeptides by digestion with almond glycopeptidase, were separated by Bio-Gel p-4 column chromatography and were purified further by thin-layer chromatography. The sugars were identified with GC-MS following the permethylation of respective oligosaccharide. To Asn-68 (NIG-65 Fc numbering (1)), two kinds of high-mannose-type oligosaccharides were bonded. To Asn-159, a kind of hybride-type and two kinds of bisected complex-type oligosaccharides were attached. From Asn-210, four kinds of bisected complex-type oligosaccharides were isolated.

A new fucosyl antigen expressed on colon adenocarcinoma and embryonal carcinoma cells.

Carbohydrate antigens on the surface of mammalian cells have recently gained renewed interest because some are specifically expressed at certain stages of cellular differentiation. Most of the useful antibodies detecting such carbohydrate antigens have been monoclonal antibodies produced by the hybridoma technique using whole cells as the immunogen or the antibodies in the sera of some of human patients. Here we report that an antiserum raised against an unusual carbohydrate linkage prepared by organic synthesis can preferentially react with certain tumour cells. Thus, an antiserum against the Fuc alpha l leads to 3Gal linkage reacted with human colon adenocarcinoma cells and murine teratocarcinoma cells but reacted only with severely restricted regions in normal tissues.