A nitrous acid procedure as a selective histochemical means of eliminating the N-sulphates of glycoconjugates

Summary A nitrous acid procedure has been shown to lead to the elimination of N-sulphates in sections of a series of tissues containing sulphated glycoconjugates. Two groups of sulphated glycoconjugate-containing tissues were used; one contained N-sulphates and other was devoid of such groupings. In the first group of tissues, mast cells of different origins and renal glomeruli in the rat were employed. Xiphoid and tracheal cartilage matrix, submandibular and sublingual gland acini and gastric, duodenal and colonic mucosae were used in the second group. Sections were treated with nitrous acid and then stained with Alcian Blue pH 1.0, high iron diamine or Aldehyde Fuchsin for sulphated glycoconjugates. Such treatment was found to diminish the staining intensities exclusively in N-sulphated glycoconjugate-containing structures such as mast cell granules and renal glomerular basement membrane, providing a means of chemically eliminating N-sulphates of glycoconjugates in tissues.     

Cytologic and histologic correlation in primary lung cancer. A study of 154 cases with resectable tumors

The accuracy of cytologic diagnosis and typing was examined in 154 patients, 113 males and 41 females, who underwent radical surgery during the past six years. There were 42 central and 112 peripheral lesions: 6 adenocarcinomas and 28 squamous-cell carcinomas were centrally located and 69 adenocarcinomas and 27 squamous-cell carcinomas were peripherally located. Repetition of sputum sampling at least three times was preferred, especially in central lesions, which were detected in 57% to 64% of the cases by either three-day-pooled or aerosol-induced specimens. Peripheral lesions required brushing to enhance the accuracy. The overall typing accuracy was 64.3%, ranging from 83.6% in squamous-cell carcinoma to 25.0% in large-cell carcinoma. Cytologic positivity correlated well with the finding of tumors more than 3 cm in diameter. Adenocarcinoma and squamous-cell carcinoma showed no significant difference in frequency of regional lymph nodal metastases. The value of judging the accuracy of cytologic diagnosis and typing on the histologic evaluation of the entire resected lesion, rather than on biopsy specimens, is emphasized.     

Induction of histidine decarboxylase activity in the spleen of mice treated with staphylococcal enterotoxin A and demonstration of its non-mast cell origin

Histidine decarboxylase (HDC) activity increased 13-, 7-, and 2-fold in the spleen, lung and liver, respectively, but not in other tissues of C57BL/6 mice injected i.v. with 50 micrograms/kg of Staphylococcal enterotoxin A (SEA). But even in the spleen, increase in the histamine level was only 1.5 times that of untreated mice. In genetically mast cell-deficient WBB6F1 - W/Wv mice HDC activity in the spleen increased to the same extent as in wild type WBB6F1 - +/+ mice on SEA treatment, but the histamine level in the spleen also increased 20-fold, whereas it increased only 1.4-fold in +/+ mice. These results suggest that the increases in HDC and histamine resulted from interaction of SEA with non-mast cells in tissues.     

Photooxidation of porphyrin in Mg-substituted horseradish peroxidase

Upon photoirradiation under aerobic conditions, the porphyrin prosthetic group in Mg-substituted horseradish peroxidase was oxidized to a mixture of its pi-cation radical and an oxidized product with an absorption band at 448 nm. The 448 nm compound was then converted to a 489 nm compound in the dark and the activation energy for the conversion was 19.3 kcal/mol. About 1 mol of O2 was consumed per mol of the 448 nm compound formed and no O2 consumption was seen in the dark reaction. The substitution of ethyl groups (meso) and hydroxyethyl groups (hemato) for the vinyl groups in protoporphyrin IX did not have an effect on the result. Under anaerobic conditions and in the presence of a suitable electron acceptor, the only photooxidation product of porphyrin was its pi-cation radical. The formation of hydroxyl radicals during irradiation under aerobic conditions was confirmed by the spin-trapping method. The formation of the above two radicals could be followed by ESR spectroscopy separately at a fixed magnetic field which was set to maximize each ESR signal. The rate of hydroxyl radical formation depended linearly on the concentration of Mg peroxidase. The photooxidation of porphyrin was slow and gave nonspecific product(s) when Mg protoporphyrin IX was present in the heme crevice of apomyoglobin or free in solution.     

Growth and aging in the rat: changes in total protein, cellularity, and polyploidy in various organs

The objectives of this study were to determine the influence of growth and aging on ploidy, cell number, and protein content of various organs. Tissue homogenates were prepared at 3, 8, 25, 50, and 100 weeks of age. Samples were analyzed for DNA per nucleus (by flow cytofluorometry), nuclei number, and protein content. Livers of 8- and 100-week-old animals were also perfused with collagenase and the released cells separated into parenchymal and nonparenchymal populations by unit gravity sedimentation. Nuclei of these cells were also analyzed for DNA. In all four zones of the kidney and in thyroid, 4n nuclei diminished in percentage between 3 and 50 weeks and increased at 100 weeks. In the growth phase these probably are cycling cells and after 50 weeks represent an increasing population of nuclei arrested after synthesis of DNA. Constant levels of ploidy were found in brain, heart, rectus abdominis, and adrenal throughout the 3-100 weeks. A dramatic increase in 4n nuclei occurred between 3 and 8 weeks in liver with little change occurring thereafter. Ploidy is a property of only parenchymal cells in liver and this probably is also true in other organs. The 4n nuclei that remain in constant proportion to the total population are established early in life and are not related to aging. They are probably tetraploid and replicate into 4n daughter cells during growth. Cerebrum shows no changes in nuclei number but exhibits a 70% increase in protein between 3 and 100 weeks. Although kidney, liver and adrenal show large increases in number of nuclei (approximately equal to fourfold) with growth, these are not as great as increases in body weight (approximately equal to 11-fold). With regard to organ protein, only liver shows increases approximating those in body weight. Increases in organ nuclei appear to occur in concert for adrenal, kidney, and liver whereas increases in organ protein bear no relationship to each other. Protein content remains at stable levels in organs of 100-week-old animals and little (adrenal, liver) or no (brain, kidney) diminution occurs in nuclei numbers.     

Increased plasma plant sterol levels in heterozygotes with sitosterolemia and xanthomatosis

Plasma sterol levels in a family of sitosterolemia and xanthomatosis were determined by a high performance liquid chromatography. Three affected siblings manifested marked xanthomatosis including subcutaneous soft tissues and generalized atherosclerosis. Two other siblings as well as children of the patients did not show such clinical symptoms and signs. Plasma levels of cholesterol, sitosterol, campesterol, and cholestanol in three affected subjects were 190 +/- 18.5, 25.9 +/- 11.6, 16.1 +/- 7.8, 1.84 +/- 0.92 mg/dl (mean +/- SD), respectively. Four daughters of the affected subjects, who should be considered as obligatory heterozygotes, showed moderately increased levels of these sterols (195 +/- 41.7, 1.33 +/- 0.44, 1.56 +/- 0.69, 0.80 +/- 0.28 mg/dl), which were significantly higher than those of normal subjects. Treatment with cholestyramine had little effect on the increased plasma plant sterol levels, but markedly decreased plasma cholestanol concentrations in two affected siblings. This report presents the clinical features of the patients with sitosterolemia and xanthomatosis and also demonstrates that heterozygotes with this disorder have increased plasma levels of plant sterols as well as cholestanol, and suggests that this rare disease might be inherited as an autosomal co-dominant trait in certain cases. The data also indicate that cholestyramine administration was not effective in this family for treatment of sitosterolemia.     

Isolation and characterization of Patnopecten mid-gut gland endo-beta-xylosidase active on peptidochondroitin sulfate

An endo-beta-xylosidase acting on the linkage region of peptidochondroitin sulfate was isolated from the mid-gut gland of the mollusc Patnopecten and purified about 375-fold, using a combination of ammonium sulfate fractionation, gel filtration on Sephacryl S-200, and DEAE-Sephacel chromatography. The pH optimum and the isoelectric point of this enzyme were 4.0 and 7.0, respectively. The molecular weight, estimated by gel filtration through Sephacryl S-200, was 78,000. The purified enzyme was completely free from protease, exoglycosidases, sulfatase, and phosphatase. This enzyme hydrolyzed the xylosyl serine linkage of the linkage region of various glycosaminoglycans, that is chondroitin sulfate, dermatan sulfate and heparan sulfate, all possessing a very small peptide segment, but not proteoglycans. It was concluded that this endo-beta-xylosidase was involved in the catabolism of proteoglycans.     

Production and Further Characterization of an Alkaline Elastase Produced by Alkalophilic Bacillus Strain Ya-B

The characteristics of the obligate alkalophilic Bacillus sp. strain Ya-B, which produces alkaline elastase extracellularly, were examined. This strain grew at pH 7.0 only in the presence of 1% or more NaCl. Its fatty acid distribution pattern was similar to that of other Bacillus species in which iso-C₁₅ and anteiso-C₁₅ were the most abundant fatty acids. About 120 mg of enzyme was recovered from 1 liter of culture broth in a medium (pH 10.1) containing mainly glucose, soymeal, and glycerol. The antiserum against this enzyme did not recognize microbial proteinases, such as subtilisins, but reacted with proteinase C, which was purified from commercial pronase. Chemical modification studies revealed that certain histidine and tyrosine residues might be involved in the enzyme activity. This enzyme underwent a partial unfolding at pHs higher than 12.0, as indicated by the circular dichroism study.