Seymour S. West

Biochemical composition and heterogeneity of heparan sulfates isolated from AH-130 ascites hepatoma cells and fluid

The glycosaminoglycan composition of AH-130 ascites hepatoma cells and fluid were examined using enzymatic digestion, electrophoresis, and sequential partition fractionation. The cell-associated glycosaminoglycans were found to consist of 93% heparan sulfate, with the remainder consisting primarily of chondroitin sulfate. The glycosaminoglycans isolated from the ascitic fluid were found to consist of 58% heparan sulfate, 26% hyaluronic acid and 16% chondroitin sulfate. Dermatan sulfate was not detected in either cells or fluid. The heparan sulfate isolated from AH-130 cells in low-sulfate and highly heterogeneous with respect to biochemical composition. Fractions isolated by partition fractionation varied from 0.14 mol sulfate/mol uronic acid to 0.6 mol sulfate/mol uronic acid. Of the total sulfate 70--80% is N-sulfate in the former and 50% in the latter. Electrophoresis in 0.1 M HCl showed a highly heterogeneous material with mobility between that of hyaluronic acid and beef lung heparan sulfate. The heparan sulfate isolated from the fluid was similar to that isolated from the cells but was, however, somewhat more homogeneous with respect to charge.

KINETICS OF FLUORESCENCE FADING OF ACRIDINE ORANGE‐HEPARIN COMPLEXES IN SOLUTION

Abstract— The irreversible photochemical fading of fluorescence of acridine orange‐heparin complexes indicates that fading involves the reaction of a “reactive” excited bound dye dimer with one in the ground state. A kinetic parameter, r“, is derived, which has a constant value over a considerable range of conditions, and which is directly related to rate constants for photophysical and photochemical processes. This parameter appears to be characteristic for heparin and may serve to identify it in the presence of other glycosaminoglycans.

Isolation and Characterization of Glycosaminoglycans from the Furth Murine Mastocytoma

New methods for isolation and fractionation by partition are described and compared with existing techniques. Substantially purer products were isolated by partition as compared to precipitation with hexadecylpyridinium chloride. The glycosaminoglycans isolated fron Furth murine mastocytoma tumor were found to consist of 78-80% heparin, 12-13% chondroitin sulfate, and 8-9% hyaluronate. Dermatan sulfate was not detected. Two heparin-like glycosaminoglycans could be isolated by partition fractionation in the phase system 1-butanol/aqueous NaCl containing hexadecylpyridinium chloride. The composition of one was typical of heparins. However, the other glycosaminoglycan contained only 0.47 moles N-sulfate/mole uronate, but had electrophoretic and partition properties characteristic of heparin.

Fluorescent Molecular Probes in Fluorescence Microspectrophotometry and Microspectropolarimetry

In recent years the biochemist and the biophysical chemist have made considerable progress in their understanding of the behavior of complex biopolymers as studied in in vitro systems. However, it is ordinarily not possible to extrapolate the results of these studies to account with entire satisfaction for the behavior of intact cells. The cytochemist has indeed been devoting considerable attention to this problem, but has been hampered by the nature of his data, which are not comparable with those produced by the biochemist and biophysical chemist. This point is well appreciated and has been explicitly discussed by Spicer, Leppi, and Stoward (1965). As a consequence, it has not been possible for the cytochemist to produce a classification of substances, detected by cytochemical means, which can be considered an accurate classification in biochemical terms. This is particularly true for the carbohydrates and mucosubstances.

PHOTOCHEMISTRY OF HEPARIN-ACRIDINE ORANGE COMPLEXES IN SOLUTION: PHOTOCHEMICAL CHANGES OCCURRING IN THE DYE AND POLYMER ON FLUORESCENCE FADING

Abstract— Photolysis of acridine‐orange‐heparin complexes in N2‐ and O2‐saturated solution results in permanent photooxidation of AO with little or no concomitant change in the heparin moiety. The major photoproduct is mono‐N‐demethylated acridine orange; in O2‐saturated solution an additional minor oxygenated product, most likely the 10‐oxide (N‐oxide) or the 9‐acridanone (acridone) is also formed. The results suggest an intermolecular electron transfer between adjacently adsorbed dye molecules. The heparin moiety plays a significant role in the photochemistry by bringing dye molecules into favorable geometric orientation for biomolecular reaction and by means of specific dye‐polymer interactions.

QUANTITATIVE MICROSCOPY IN BACTERIOLOGY

One way to study bacteria, when present in small numbers and in low concentration, is to employ some form of scanning microscopy. The microscope must be employed both to resolve individual bacteria and to deal with the extremely small quantities of chemical substances which they contain. It is also apparent that some form of scanning must be employed if very small numbers of organisms are to be dealt with effectively on an automatic and continuous basis. Scanning instrumentation can be addressed to a variety of problems in bacteriology. In all instances it is possible to obtain a count of the number of organisms present. It is also possible to record data which is representative of additional information, as well as count. A few general examples are:

Calibration of a microspectrofluorophotometer in absolute units of radiation.

Methods are described for direct calibration of a microspectrofluorophotometer in absolute units of radiation. The direct calibration was checked by determining the quantum yields of quinine bisulfate and rhodamine B solutions and found to give results within 7% of published values. Methods for calibrating a phosphor particle standard for day to day use are also described.

Intravesical CDDP therapy compared with combined CDDP and external radiation in noninvasive bladder cancer Monitored with quantitative fluorescence cytology

Results of intravesical CDDP or CDDP combined with external beam radiation are compared in a group of 13 patients with low-stage bladder cancer. Six patients with low-stage bladder cancer received 4 or 12 treatments of CDDP intravesically with an initial complete response in 3 patients. Within six months, recurrent disease developed in 2 of 3 patients. Seven patients received the combination therapy of 400 rad (weekly for six weeks) followed two hours later with 50 mg of intravesical CDDP. A positive response was observed initially in all 7 patients as determined by pathology, PAP cytology, fluorescence cytology, and quantitative nuclear fluorescence determinations. Therapy was discontinued in 1 patient in each group because of irritative symptoms. The results indicate combination therapy is of tolerable toxicity, and quantitative fluorescence cytology is a useful adjuvant for guiding future treatments in patients with low- and high-grade bladder tumors.

Chemical and Cytochemical Studies of Heparan Sulfates from AH-130 Ascites Hepatoma

Publisher Summary AH-130 is a transplantable ascites hepatoma derived from a solid hepatoma induced in rats by an azo dye. This chapter discusses the chemical and cytochemical studies of sulfates from AH-130 ascites hepatoma. In the study, the tumor cells were maintained intraperitoneally in Sprague-Dawley rats. The average survival span of the tumor-bearing rats was nine days. Glycosaminoglycans (GAG) were isolated from AH-130 cells, ascitic fluid, and normal rat liver by Pronase digestion, treatment with trichloroacetic acid, and precipitation with hexadecylpyridinium chloride. The chapter lists the yield of total GAG from AH-130 and normal rat liver. AH-130 cells contain approximately six times as much GAG as normal rat liver.