Cross-reaction of synthetic alpha-(1----3)-branched glucans with rabbit anti-N4 dextran.

Cross-reactions of four synthetic branched glucans (3-O-alpha-D-glucopyranosyl-(1----6)-alpha-D-glucopyranans: V39, V17, V37, and V32, each containing one unit glucose branches amounting to 11-12%, 33-43%, 50-54%, and 71-100%, respectively) with rabbit anti-N4 dextran were examined. All four samples precipitated antibodies raised in rabbits by injecting N4 dextran-concanavalin A conjugate. The ability of glucans to precipitate antibody depended on the quantity of branches, samples with more branches precipitating less antibody nitrogen under the same conditions. This may indicate an inhibitory effect of the branches on precipitation. Oligosaccharide inhibition assay showed that the precipitation reactions were specific for (1----6)-alpha-D-glucopyranosyl linkages, and the maximum size of the alpha-(1----6)-specific antibody combining site corresponded to isomaltopentaose. Determination of antibody nitrogen and glucan in the precipitates indicated that the ratios of one combining site of antibody to numbers of glucose residues were 1:9 (V39), 1:11 (V17), and 1:16 (V37) in the extreme antibody excess region. A synthetic sample of manno-glucan ((1----6)-alpha-D-glucopyranan containing about 27% of randomly linked 3-O-alpha-D-mannopyranosyl side chains) also reacted with the same antibody.

DNA content as a prognostic factor in endometrial carcinoma.

Two hundred thirty-three cases of endometrial carcinoma were analyzed for DNA content using flow cytometry of cell nuclei extracted from archival paraffin blocks. The median follow-up time for the cases was 8.7 years. Aneuploidy, present in 18% of tumors overall, was associated with adverse histologic type, high grade, and depth of invasion in the uterus. Aneuploidy was not detected in low-grade carcinomas. A DNA index greater than 1.5 strongly predicted death from disease. For endometrial adenocarcinoma and papillary serous carcinoma, this finding appeared independent of stage or tumor grade. The percentage of cells in S phase or G2 + M of the cell cycle did not predict clinical outcome in diploid tumors. Application of DNA analysis to low-stage endometrial cancers of high grade or of papillary serous type may be useful for selecting a subgroup of patients for adjuvant therapy.

The synthesis of antigenic determinants for yeast D-mannans and a linear (1to 6)-alpha-D-gluco-D-mannan, and their protein conjugates.

2-O-Benzoyl-3,4,6-tri-O-benzyl-1-O-tosyl-D-mannopyranose and 2,3,4-tri-O-benzyl-6-O-(N-phenylcarbamoyl)-1-O-tosyl-D-glucopyranose were allowed to react with partially blocked 2-[4-(ptoluensulfonamido)phenyl]ethyl alpha-D-manno- and gluco-pyranosides. Disaccharides having alpha-D-Manp-(1 to 2)-alpha-D-Manp, alpha-D-Manp-(1 to 6)-alpha-D-Glcp, alpha-D-Manp-(1 to 6)-alpha-D-Manp, and alpha-D-Glcp-(1 to 6)-alpha-D-Manp structures, and a branched trisaccharide having the structure alpha-D-Manp-(1 to 2)-[alpha-D-Manp-(1 to 6)]-alpha-D-Manp were synthesized. The oligosaccharid:s were deblocked with sodium in liquid ammonia to give glycopyranosides having a free primary aromatic amine which were converted into isothiocyanate derivatives with thio-phosgene. The functionalized oligosaccharides were then coupled to bovine serum albumin to give protein conjugates.

The synthesis of alpha and beta-(1 less than 2) and-(1 less than 3)-linked glucopyranose disaccharides and their protein conjugates.

2,3,4-Tri-O-benzyl-6-O-(N-phenylcarbamoyl)-1-O-tosyl-D-glucopyranose and 3,4,6-tri-O-benzyl-2-O-p-nitrobenzoyl-1-O-tosyl-D-glucopyranose were allowed to react with partially blocked 2-[4-p-toluenesulfonamido) phenyl]ethyl alpha- and beta-D-glucopyranosides. Disaccharides having the structure alpha-D-Glcp-(1 goes to 2)-alpha-D-Glcp, alpha-D-Glcp-(1 goes to 3)-alpha-D-Glcp, beta-D-Glcp-(1 goes to 2)-beta-D-Glcp, and beta-D-Glcp-(1 goes to 3)-alpha-D-Glcp were synthesized. The oligosaccharides were debenzylated with sodium in liquid ammonia to give disaccharides having a free primary aromatic amino group, which were converted into isothiocyanate derivatives and then coupled to various proteins to give the corresponding conjugates.

Studies on the synthesis of propyl 4-O-beta-D-galactopyranosyl-alpha-D-galactopyranoside.

2-O-Benzoyl-3,6-di-O-benzyl-4-O-(chloroacetyl)-, 4-O-acetyl-2-O-benzoyl-3,6-di-O-benzyl-, and 2-O-benzoyl-3,4,6-tri-O-benzyl-alpha-D-galactopyranosyl chloride were converted into the corresponding 2,2,2-trifluoroethanesulfonates, and these were treated with allyl 2-O-benzoyl-3,6-di-O-benzyl-alpha-D-galactopyranoside, to give allyl 2-O-benzoyl-4-O-[2-O-benzoyl-3,6-di-O-benzyl-4-O-(chloroacetyl)- beta-D-galactopyranosyl]-3,6-di-O-benzyl-alpha-D-galactopyranoside (26; 41% yield), allyl 4-O-(4-O-acetyl-2-O-benzoyl-3,6-di-O-benzyl-beta-D- galactopyranosyl)-2-O-benzoyl-3,6-di-O-benzyl-alpha-D-galactopyranoside (27; 62% yield), and allyl 2-O-benzoyl-4-O-(2-O-benzoyl-3,4,6-tri-O-benzyl-beta-D- galactopyranosyl)-3,6-di-O-benzyl-alpha-D-galactopyranoside (28; 65% yield). All disaccharides were free from their alpha anomers. Disaccharides 26 and 27 were found to be base-sensitive, and were de-esterified by KCN in aqueous ethanol, and debenzylated with H2-Pd. Attempts to produce (1----4)-beta-D-galactopyranosides from the coupling of a number of fully esterified D-galactopyranosyl sulfonates to allyl 2,3,6-tri-O-benzoyl-alpha-D-galactopyranoside were unsuccessful.

Synthesis and characterization of propyl O-beta-D-galactopyranosyl-(1----4)-O-beta-D-galactopyranosyl-(1----4)- alpha-D-galactopyranoside.

Allyl 4-O-(4-O-acetyl-2-O-benzoyl-3,6-di-O-benzyl-beta-D-galactopyranosyl)- 2-O-benzoyl-3,6-di-O-benzyl-alpha-D-galactopyranoside was O-deallylated to give the 1-hydroxy derivative, and this was converted into the corresponding 1-O-(N-phenylcarbamoyl) derivative, treatment of which with dry HCl produced the alpha-D-galactopyranosyl chloride. This was converted into the corresponding 2,2,2-trifluoroethanesulfonate, which was coupled to allyl 2-O-benzoyl-3,6-di-O-benzyl-alpha-D-galactopyranoside, to give crystalline allyl 4-O-[4-O-(4-O-acetyl-2-O-benzoyl-3,6-di-O-benzyl- beta-D-galactopyranosyl)-2-O-benzoyl-3,6-di-O-benzyl-beta-D-galactopyran osyl]- 2-O-benzoyl-3,6-di-O-benzyl-alpha-D-galactopyranoside (15) in 85% yield, no trace of the alpha anomer being found. The trisaccharide derivative 15 was de-esterified with 2% KCN in 95% ethanol, and the product O-debenzylated with H2-Pd, to give the unprotected trisaccharide. Alternative sequences are discussed.

Activity of branched dextrans in the acceptor reaction of a glucosyltransferase (GTF-I) from Streptococcus mutans OMZ176.

The ability of several native and chemically synthesized, branched dextrans to stimulate the activity of an alpha-D-glucosyltransferase (GTF-I) of Streptococcus mutans has been compared. The enzyme catalysed the transfer of glucosyl residues from sucrose with the formation of water-insoluble (1----3)-alpha-D-glucan. The rate of this reaction was greatly increased in the presence of dextran, and the extent of stimulation was negatively correlated with the degree of branching of the added dextran. The results refute the concept that growth of water-insoluble glucan occurs from the multiple, non-reducing termini of dextran acceptors.

Steric control in the polymerization of 1,2-anhydro-3,4,6-tri-O-benzyl-beta-D-mannopyranose.

Polymerization of 1,2-anhydro-3,4,6-tri-O-benzyl-beta-D-mannopyranose under acid catalysis has led to a series of polymers varying in anomeric configuration from approximately 90% alpha to 70% beta. Optical rotations follow 13C-n.m.r. estimates of anomeric composition linearly over this range. Low-temperature polymerization with trifluoromethanesulfonic anhydride as initiator favors mainly cis-opening of the anhydro ring, presumably through the intermediary of a macroester. These results are compared with related glycosylation and polymerization reactions on 1,2-anhydro sugar derivatives, and some mechanistic conclusions are proposed.

Regioselective alkylation and acylation of carbohydrates engaged in metal complexes.

Substituted carbohydrate derivatives (D-gluco, D-manno, and D-galacto) having two free hydroxyl groups were converted into their metal chelates by reaction with sodium hydride and a metal chloride (cupric or mercuric) in either oxolane or 1,2-dimethoxyethane. Reaction of these metal chelates with allyl, benzyl, or methyl iodide, or acetic anhydride or benzoyl chloride, gave a mixture of di-, mono-, and un-substituted products in which one monosubstituted derivative preponderated. In the alkylation of copper chelates, disubstitution was absent. Appropriate choice of metal ion and organic reagent often permits selective substitution on either of two hydroxyl groups. Products were separated by liquid chromatography, and characterized by 1H- and 13C-n.m.r. spectroscopy.

Lymphocyte subsets and activation antigens in a reference population: a flow cytometric study using single and double antibody staining.

The transferrin receptor (TR), the HLA DR antigen (DR), and the antigen binding OKT10 (T10) are present on activated lymphocyte populations. The authors have studied their expression and that of antigens defined by eight commercial monoclonal antibodies on peripheral blood lymphocytes of 50 healthy hospital workers aged 23-60 years. A whole blood lysis technique was employed and cells were enumerated on a flow cytometer. The percentage of cells bearing the three activation antigens were generally low: mean values for T10 being 7.2%; for TR 1.8%; and for DR 8.8%. There was, however, considerable variability, with occasional subjects having 20% or more cells positive for one of the three antigens. High values of one activation antigen did not correspond with high values of another in the same subject. Nor was there correlation of the presence of activation antigens with the occurrence of cells bearing T11, T4, T8, Leu 1, Leu 2a, Leu 3a, or Leu 7. Double labelling with the following pairs of fluorescein (FITC) and phycoerythrin (PE) labeled antibodies: Leu 2a, DR; Leu 3a, DR; Leu 3a, Leu 2a; TR, DR, indicated that simultaneous expression of the corresponding antigens do not normally occur on lymphocytes of healthy individuals. Double labelling with B1 and DR in five subjects indicated the presence of B1 (+) DR (-) cell population. No pattern of relationship could be detected among common clinical variables or HLA type and an increased expression of activation antigens.

Papillary-cystic neoplasm of the pancreas.

Papillary-cystic neoplasm of the pancreas is a rare tumor which has recently been described as a distinct, clinicopathological entity. Forty-four previously described cases from the literature and an additional 52 cases referred to in a brief abstract are reviewed. An additional case occurring in a 25-year-old woman is reported. This tumor occurs primarily in young women (mean age 23.3 years) and presents as a large abdominal mass (mean size 10.3 cm) in 63% of patients. Treatment is surgical resection of the involved portions of the pancreas and results of treatment are excellent following this form of therapy alone. The tumor appears to represent a low-grade malignancy, possibly of ductal cell origin. Only two patients with recurrences and two patients with metastatic disease have been reported. Surgeons should be aware of this curable pancreatic malignancy as they are most often responsible for the evaluation and treatment of the patient presenting with a large abdominal mass.

Lethal effect of neutral mannan fraction of bakers' yeast in mice.

A simple polysaccharide, the neutral mannan from Saccharomyces cerevisiae wild type strain (WNM) was found to kill ddY strain mice by intravenous administration, showing a LD50 value of 12.2 mg/kg. On the other hand, the acidic mannan fraction from the same yeast containing phosphate (WAM025), and chemically phosphorylated WNM (WNM-P) were practically non-toxic. Concerning the relationship between chemical structure and lethal effect of these mannans, it was demonstrated that a mannan possessing a highly branched structure exhibited stronger lethality than those with less branched structures. Against C3H/HeJ strain mice with no responsiveness to lipopolysaccharide, the LD50 value of WNM was as high as 75 mg/kg. Pretreatment with 500 mg/kg of D-mannose, N-acetyl-D-glucosamine, D-galactose, and L-fucose prevented mice from the lethal effects of WNM. However, WNM (LD100) did not show any lethal effect in mice for 2 to 12 hr after treatment with dexamethasone, an anti-inflammatory steroid.

Paget's disease of the vulva: prevalence of associated vulvar adenocarcinoma, invasive Paget's disease, and recurrence after surgical excision.

OBJECTIVE: Our aim was to determine the prevalence of associated vulvar adenocarcinoma, invasive Paget's disease, and recurrence of Paget's disease of the vulva. STUDY DESIGN: A retrospective review of tumor and pathology registries at 8 institutions is presented. Patients with recurrent disease were excluded. Histologic slide review was performed. RESULTS: The median age of the 100 patients was 70 years. The median duration of pruritus before surgery was 2 years. Thirty-four percent of patients experienced a recurrence at a median of 3 years. There was a 12% prevalence of invasive vulvar Paget's disease and a 4% prevalence of associated vulvar adenocarcinoma. One patient died of Paget's disease with associated vulvar adenocarcinoma. CONCLUSIONS: Paget's disease of the vulva is rarely associated with an underlying vulvar adenocarcinoma or invasive Paget's disease, but there is a high recurrence rate.

Immunochemical similarity of synthetic alpha-(1----3)-branched D-glucans to dextran B1375.

Anti-dextran B1375 antibodies were raised in rabbits by injecting formalin-killed Leuconostoc mesenteroides strain NRRL B1375, and the anti-dextran serum was used to examine native dextran B1375, and synthetic linear and four alpha-(1----3)-branched alpha-(1----6)-D-glucopyranans for similarities. The antiserum reacted with the homologous dextran B1375 and also with all the synthetic linear and branched glucans. Precipitation and precipitation-inhibition studies indicated that the antiserum contained at least three groups of antibodies with different specificities, the first specific for linear alpha-(1----6)-D-glucopyranan structure, the second specific for alpha-D-glycopyranosyl-(1----3)-branching and the last specific for another, unknown structure present in the dextran B1375 molecule. Two samples of the synthetic branched glucans were shown to be immunochemically the most similar to natural dextran B1375 by inhibition experiments.

Immunochemical studies of the mannans of Saccharomyces cerevisiae X2180-1A-5 and Saccharomyces cerevisiae 4484-24D-1 mutant strains, with special reference to their phosphate content.

The mannans from Saccharomyces cerevisiae mutant strains X2180-1A-5 and 4484-24D-1, both of which were shown to contain small amounts of phosphate (less than 0.2%), were fractionated on a column of diethylaminoethyl-Sephadex into five subfractions designated as fractions I to V. These subfractions contain different amounts of phosphate, ranging from 0.03 to 0.09 (strain X2180-1A-5) and from 0.01 to 0.17% (strain 4484-24D-1). Fractions I to IV from strain X2180-1A-5 showed nearly identical precipitin activities against the homologous anti-whole cell serum, whereas fraction V, containing the largest amount of phosphate and protein among this mannan subfraction series, showed unexpectedly weaker precipitin activity than those of the other fractions. A synthetic mannan consisting or consecutive alpha-1 leads to 6-linked D-mannopyranosyl residues was found to be cross-reactive with all the mannan subfractions of strain X2180-1A-5 against anti-X2180-1A-5 serum. On the other hand, antibody-precipitating activities of the mannan subfractions of the latter strain were proportional to their phosphate content, although the increments of precipitated antibody nitrogen among the subfractions were quite small. However, fraction V of this mannan subfraction series, containing the largest amounts of phosphate and protein, showed lower precipitin activity than did the other four fractions. These findings indicate that mannans containing no phosphate or relatively small amounts of phosphate, such as those investigated in the present study, are less heterogeneous in the densities of the branching moieties than are highly phosphorylated mannans. These findings suggest that the transfer step of mannosyl-1-phosphate into the precursor(s) of the wild-type strain mannans during the biosynthetic process corresponds to the key reaction responsible for the anionic heterogeneity due to the density heterogeneity of the antigenic determinants.

Marked hyperamylasemia associated with carcinoma of the lung.

We present a case of marked hyperamylasemia associated with undifferentiated small cell carcinoma of the lung. The serum alpha-amylase had an electrophoretic mobility similar to that of salivary gland enzyme (s-type), and that in tissue extracts from the lung tumor and metastatic skin nodules showed similar migration. No evidence of a salivary or pancreatic cause of hyperamylasemia was found at autopsy, and macroamylasemia was excluded. We suggest that the strikingly increased serum alpha-amylase activity was a result of ectopic production of this enzyme by tumor. We compare results for this case to other published reports.

A pathologic study of benign breast diseases in Tokyo and New York.

Two hundred thirty-two biopsies of benign breast conditions from the National Cancer Center Hospital in Tokyo and 263 comparable biopsies from Memorial Hospital in New York City were reviewed. The component lesions of benign breast disease were identified and evaluated according to their relative frequency for different ages. Apocrine cysts, apocrine hyperplasia, intraductal hyperplasia, sclerosing adenosis, blunt duct hyperplasia, and atypical lobular hyperplasia were at least twice as common in biopsies from American women. Solitary papillomas were twice as common among biopsies from Japanese patients and tended to show more epithelial proliferation and sclerosing papillomatosis than did Americans. No major differences were found in the frequency of cysts, duct stasis, periductal mastitis, sclerosing intraductal papillomatosis, fibroadenomatoid mastopathy or fibroadenomas. Assuming intraductal hyperplasia and atypical lobular hyperplasia may represent premalignant epithelial changes, the high frequency of these lesions in New York biopsies when compared to Tokyo biopsies correlates well with the higher rate of breast cancer in the United States as compared to Japan. The greater frequency of lobular proliferative lesions and apocrine disease in New York suggests that these lesions may be produced by factors which also predispose American women toward breast cancer.