neu is not involved in N-[4-(5-nitro-2-furyl)-2-thiazolyl]-formamide-induced bladder carcinoma or 2-amino-4-(5-nitro-2-furyl)thiazole transformation of rat bladder epithelial cells.

Enhanced c-erbB-2/neu expression has been linked with a poor prognosis in human bladder cancer. Previous reports have shown that a point mutation at nucleotide T2012 in the coding region of the transmembrane domain of the rat gene is sufficient to confer transformation potential on this gene. We examined the comparative levels of p185neu as well as the sequence around the hotspot (T2012) of the neu gene of rat bladder cells transformed by 2-amino-4-(5-nitro-2-furyl)thiazole (ANFT) or established in culture from N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (FANFT)-induced rat bladder tumors. We concluded that increased p185neu expression did not correlate significantly with tumorigenicity. No alterations in nucleotide sequences of the neu gene were observed in either in vitro model.

Neu is not involved in N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide- induced bladder carcinoma or 2-amino-4-(5-nitro-2-furyl)thiazole transformation of rat bladder epithelial cells.

Enhanced c-erbB-2/neu expression has been linked with a poor prognosis in human bladder cancer. Previous reports have shown that a point mutation at nucleotide T2012 in the coding region of the transmembrane domain of the rat gene is sufficient to confer transformation potential on this gene. We examined the comparative levels of p185neu as well as the sequence around the hotspot (T2012) of the neu gene of rat bladder cells transformed by 2-amino-4-(5-nitro-2-furyl)thiazole (ANFT) or established in culture from N-[-4-(-5-nitro-2-furyl)-2- thiazolyl]formamide (FANFT)-induced rat bladder tumors. We concluded that increased p185neu expression did not correlate significantly with tumorigenicity. No alterations in nucleotide sequences of the neu gene were observed in either in vitro model.

A comparison of the effects of sodium saccharin in NBR rats and in intact and castrated male F344 rats.

High doses of sodium saccharin (NaSac) increase proliferation in the bladder of the rat, with a male preponderance. The possibility that alpha 2u-globulin is involved in its mechanism of action was evaluated by feeding it at 7.5% of the diet to NCI-Black-Reiter (NBR) male rats, which do not synthesize liver-derived alpha 2u-globulin. NaSac affected urinary parameters similarly in F344 and NBR male rats, but NBR rats consumed more water leading to greater urinary volume. NaSac produced less proliferation in NBR than in intact F344 rats, with intermediate changes in castrated F344 males, which had intermediate urinary alpha 2u-globulin levels.

Vasoactive intestinal peptide hydrolysis by antibody light chains.

This paper describes evidence for hydrolysis of a neuropeptide, vasoactive intestinal peptide (VIP), by light chains purified from the IgG of a human subject positive for VIP binding antibodies. Purified IgG was digested with papain, resultant fragment antigen binding (Fab) fragments were reduced with 2-mercaptoethanol and alkylated with iodoacetamide, and light chains were purified by chromatography on immobilized antibodies to light chains and immobilized antibodies to heavy chains. Non-immunoglobulin components were undetectable in the light chain preparation, judged by sodium dodecyl sulfate-electrophoresis and Western blotting with anti-heavy and anti-light chain antibodies. The light chains hydrolyzed VIP with specific activity 32-fold greater than that of Fab, the pH optimum for light chain-mediated VIP hydrolysis was 7.0-7.5, and the hydrolytic activity was saturable (Vmax, 0.19 pmol/min/microgram light chains; substrate concentration at Vmax/2,380 nM).

Cleavage of vasoactive intestinal peptide at multiple sites by autoantibodies.

Vasoactive intestinal peptide (VIP) fragments generated by autoantibodies purified from the blood of two human beings were separated and sequenced. Based on the identity of these fragments, seven peptide bonds cleaved by the antibodies were identified. Six of the seven scissile bonds are clustered in the region of VIP spanning residues 14-22 and were cleaved by antibodies from both human subjects. The seventh scissile bond is located at residues 7-8 and was cleaved by antibodies from one of the subjects. The scissile bonds link amino acid residues with different size, charge, and hydrophobicity. The hydrolytic activity of the antibodies was selective in that they failed to hydrolyze polypeptides unrelated in sequence to VIP (insulin and atrial natriuretic peptide). These observations demonstrate substrate specific hydrolysis by naturally occurring antibodies and expand the range of peptide bonds hydrolyzed by these antibodies.

Characterization of beta-glucosidases with high specificity for the cyanogenic glucoside dhurrin in Sorghum bicolor (L.) moench seedlings.

Two beta-glucosidases exhibiting high specificity for the cyanogenic glucoside dhurrin have been purified to near homogeneity from seedlings of Sorghum bicolor. Dhurrinase 1 was isolated from shoots of seedlings grown in the dark. Dhurrinase 2 was isolated from the green shoots of young seedlings grown in the light. The two enzymes were similar in following characteristics: their optimum activity is around pH 6.2; the enzymes are stable above pH 7; they are effectively inhibited by the beta-glycosidase inhibitors nojirimycin delta-gluconolactone and 1-amino-beta-D-glucoside. On the other hand, they clearly differed in other properties, e.g., molecular weights, isoelectric points, and substrate specificity. Moreover, dithiothreitol has no effect on dhurrinase 1, but is necessary for the activity of dhurrinase 2. Preliminary investigations indicate that the two enzymes are located in different parts of the sorghum seedlings: dhurrinase 1 is found in the coleoptiles and hypocotyls; dhurrinase 2 occurs in the leaves. Dhurrin (p-hydroxy-(S)-mandelonitrile-beta-D-glucoside) and its structural analog without the hydroxyl group, sambunigrin, were the only substrates hydrolyzed at high rate, the Km values with both enzymes being 0.15 and 0.3 mM, respectively. All other cyanogenic glucosides tested, as well as synthetic substrates such as 4-nitrophenyl-beta-D-glucoside, were in general poor substrates, especially for dhurrinase 1, the enzyme isolated from coleoptile and hypocotyl tissue. Dhurrinase 1 appears to exist within the seedlings as a tetramer (Mr - 2-2.4 X 10(5)) which dissociates without loss of activity into a dimeric form (Mr = 1-1.1 X 10(5)) upon extraction and purification. There is only one monomeric subunit with Mr = 5.7 X 10(4). Isolectric focusing and chromatofocusing of purified dhurrinase 1 showed the presence of at least three isomeric forms, but their relationship to each other is not known at the present time. Dhurrinase 2 appears to be a tetrameric protein with Mr = 2.5-3 X 10(5); it also has only one monomeric subunit of Mr = 6.1 X 10(4). In contrast to many other beta-glucosidases, the dhurrinases are not glycoproteins.

Milligram to gram scale purification and characterization of dextransucrase from Leuconostoc mesenteroides NRRL B-512F.

A sequence of dextranase treatment, DEAE-cellulose chromatography, affinity chromatography on Sephadex G-200, and chromatography on DEAE-Trisacryl M has been optimized to give a dextransucrase preparation with low carbohydrate content (1-100 micrograms/mg protein) and high specific activity (90-170 U/mg protein) relative to previous procedures, in 30-50% yield. Levansucrase was absent after DEAE-cellulose chromatography, and dextranase was undetectable after Sephadex G-200 chromatography. The method could be scaled up to produce gram quantities of purified enzyme. The purified dextransucrase had a pH optimum of 5.0-5.5, a Km of 12-16 mM, and produced the same lightly branched dextran as before purification. The purified enzyme was not activated by added dextran, but the rate of dextran synthesis increased abruptly during dextran synthesis at a dextran concentration of approximately 0.1 mg/mL. The enzyme had two major forms, of molecular weight 177,000 and 158,000. The 177,000 form predominated in fresh preparations of culture supernatant or purified enzyme, whereas the amount of the 158,000 form increased at the expense of the 177,000 form during storage of either preparation.

Relative, quantitative effects of acceptors in the reaction of Leuconostoc mesenteroides B-512F dextransucrase.

The acceptor reaction of dextransucrase consists of the transfer of D-glucosyl groups from sucrose to other carbohydrates, and occurs at the expense of dextran synthesis. In the present study, solutions of [14C]sucrose and of each of seventeen acceptor sugars were digested with highly purified Leuconostoc mesenteroides B-512F dextransucrase. The products were separated by paper chromatography, and quantitated by liquid scintillation counting. Maltose was the most effective acceptor; its products, members of an isomaltodextrinyl-maltose series (d.p. 3 to 6), accounted for greater than 75% of the D-glucosyl groups of sucrose. Other acceptors giving rise to a similar series of oligosaccharide products were (in order of decreasing effectiveness): isomaltose, nigerose, methyl alpha-D-glucoside, 1,5-anhydro-D-glucitol, D-glucose, turanose, methyl beta-D-glucoside, cellobiose, and L-sorbose. Lactose, raffinose, melibiose, D-galactose, and D-xylose each gave a single, mono-D-glucosylated product; D-fructose and D-mannose each gave a pair of mono-D-glucosylated (disaccharide) products. Another series of digests contained sucrose and various proportions of maltose. As the level of maltose increased, the size of the largest oligosaccharide acceptor-product decreased, and less dextran was produced. The virtual absence of high-d.p. (8 to 13) oligosaccharide products in all acceptor digests is interpreted as evidence against a role for acceptors as primers of dextran synthesis.