Zbigniew Walaszek

Conformations of the d-glucarolactones and d-glucaric acid in solution☆

Abstract The conformations of d -glucaric acid ( 1 ), d -glucaro-1,4-lactone ( 2 ), d -glucaro-6,3-lactone ( 3 ), and d -glucaro-1,4:6,3-dilactone ( 4 ) in solution were investigated by 1 H-n.m.r. and 13 C-p.F.t., n.m.r. spectroscopy. The solvents used were deuterium oxide, methanol- d 4 , and dimethyl sulfoxide- d 6 , and praseodymium chloride was employed as a lanthanide shift-reagent. For 2 , it was found that the conformational equilibrium 3 E ( d ) E 3 ( d ) exists in solution, and that the OH-5 group tends to occupy the position over the lactone ring in the favored E 3 ( d ), gg conformation. The n.m.r. data for 3 indicated that the conformational equilibrium is shifted in favor of the 4 E ( d ) E 4 ( d ), gt conformation in solution. The dienvelope conformation 3 E : E 4 ( d ) was found to be the favored conformation of 4 . For 1 , a conformational equilibrium between one planar, zigzag form and two sickle forms was indicated by the n.m.r. data observed. 13 C-N.m.r. spectroscopy proved to be a convenient method for monitoring the lactonization of 1 , and the hydrolysis of its lactones. Lactones other than 2 – 4 were not found in solutions prepared from 1 – 4 , either during their mutarotation or after equilibration at 30°.

Conformational studies on aldonolactones by n.m.r. spectroscopy. Conformations of d-glucono-1,5-lactone and d-mannono-1,5-lactone in solution

Abstract The conformations of d -glucono-1,5-lactone (1) and d -mannono-1,5-lactone (2) in solution were investigated by 1H- and 13C-n.m.r. spectroscopy. Conformational equilibria for 1 and 2 were found to lie strongly in favor of the 4H3( d ),gg and B2,5( d ),gg conformations, respectively.

Dietary glucarate-mediated reduction of sensitivity of murine strains to chemical carcinogenesis

Serum beta-glucuronidase activity is shown to differ quantitatively in the following strains of mice, listed in order of increasing activity: C3H, C57BL/6 less than BALB/c, DBA/2, ICR less than SENCAR, A/He. The level of the enzyme in the murine strains is shown to correlate with the urinary excretion of 17-ketosteroids, which in turn reflects the endogenous level of androgens. Dietary calcium D-glucarate, an in vivo beta-glucuronidase inhibitor, reduced the steady state level of both beta-glucuronidase and 17-ketosteroid excretion in the highly susceptible A/He and SENCAR strains to that of strains known to be resistant to chemical carcinogenesis. Sensitivity of the A/He strain is significantly reduced by dietary calcium glucarate, which is shown to inhibit DNA binding and the induction of pulmonary adenomas by benzo[a]pyrene.

Conformational studies on aldonolactones by N.M.R. spectroscopy. Conformations of d-pentono-1,4-lactones in solution

Abstract The conformations of d -pentono-1,4-lactones in solution were studied by 1 H- and 13 C-n.m.r. spectroscopy. Conformational equilibria between the 3 E and E 3 forms were found to favor, strongly, that having the OH-2 group quasiequatorially oriented. The exocyclic, CH 2 OH groups in these lactones generally favor the gauche-gauche disposition around the C-4-C-5 bond, except for d -lyxono-1,4-lactone, which favors the trans-gauche arrangement.

An oncofetal 60-kilodalton protein in the plasma of tumor-bearing and carcinogen-treated rats

A Mr 60,000 protein, detected by its ability to induce the release of RNA from isolated nuclei, is present in the plasma of tumor-bearing and carcinogen-treated rats, together with low amounts of 2 messenger RNA transport proteins identified earlier in normal cells. The Mr 60,000 protein has been identified in tumor cell cytoplasm and in amniotic fluid, but does not appear to cross the placental barrier. Significant amounts of the Mr 60,000 oncofetal protein appear in the plasma of carcinogen-treated rats within a few weeks of treatment. It may be the fetal form of the adult messenger RNA transport proteins.

Repression by sustained-release β-glucuronidase inhibitors of chemical carcinogen-mediated induction of a marker oncofetal protein in rodents

The degree of induction of an oncofetal protein marker in rodents by selected chemical carcinogens has been correlated with changes in carcinogenicity induced by dietary D-glucaro-1,4-lactone (GL) based anticarcinogens. These potent anticarcinogens may act to increase the clearance of carcinogens as glucuronides through the inhibition of beta-glucuronidase. The sustained-release forms are particularly effective, 1.5 mmol/kg of GL maintaining serum beta-glucuronidase activity at or below 50% for only 1 h, while an equivalent amount of calcium glucarate (CGT) maintained this level of inhibition for over 5 h. CGT or other sustained-release inhibitors, when fed to rodents during administration of carcinogens that undergo glucuronidation, caused a marked reduction in the induction of the marker protein. For those systems where other markers of carcinogenesis were also assessed, it was determined that the inhibition of marker-protein induction was quantitatively similar to both the inhibition of binding of the carcinogen to DNA and the subsequent induction of tumors in target organs.

Effects of calcium glucarate on the promotion of diethylnitrosamine-initiated altered hepatic foci in rats

Calcium glucarate (CGT), an inhibitor of beta-glucuronidase, is a potent inhibitor of chemically-induced tumors when administered orally. The present study was undertaken to determine the effects of CGT on the promotion of hepatocarcinogenesis by phenobarbital following initiation with diethylnitrosamine (DENA). Partially hepatectomized, DENA-initiated female Sprague-Dawley rats, previously maintained only on chow diet for 2 months, were supplemented with either 0.05% phenobarbital alone or 0.05% phenobarbital plus 4% dietary CGT, for varying time intervals up to 6 months. Histopathologic evaluation of the liver sections showed that CGT significantly delayed the development of altered hepatic foci (AHF). By the seventh month post-initiation, however, the frequency and severity of changes seen in the livers of experimental animals approximated those of the controls.

β-Glucuronidase levels in patients with fibrocystic breast disease

Certain enzymes in tissues and body fluids may, through reversal of the detoxification process, influence the composition and availability of steroid hormones, toxins, and carcinogens. The ubiquitous enzyme β-glucuronidase, which hydrolyzes glucuronide conjugates, thereby reversing one of the main detoxification and excretion pathways, was found to vary in concentration in different cysts over a 300-fold range. The distribution was a continuum, devoid of discrete sub-populations. Evidence obtained on selected cyst fluids of high and low β-glucuronidase activities indicated that the level of the enzyme significantly influenced the ratio of unconjugated: glucuronidated estradiol. The patients with fibrocystic breast disease fell into 2 distinct subpopulations on the basis of their serum β-glucuronidase activity. In one group the activity was near normal, while in the second group the average serum β-glucuronidase activity was 3-fold higher than in the women who did not have benign breast disease.

Characterization of a 60,000-dalton Oncofetal Protein from the Plasma of Tumor-Bearing Rats

A tumor cell-associated protein, previously shown to be present in the circulation of carcinogen-treated and tumor-bearing animals and cancer patients, has now been identified in the cytosol of embryonic tissue. This oncofetal protein, which is absent from the plasma of normal animals, has been purified from the plasma of tumor-bearing rats by a series of steps including ammonium sulfate fractionation and chromatography on Sepharose CL-6B and on CM Affi-Gel Blue. The tumor and fetal-associated 60-kd rat factors appear to be identical based on their reactivity to polyclonal antibody produced against the tumor factor. The factor, assayed by its ability to induce the transport of RNA from isolated nuclei, is a phosphoprotein with a minimum molecular weight of 60,000, as determined by polyacrylamide gel electrophoresis. In its purified form it is phosphorylated in the presence of the catalytic subunit of heart muscle protein kinase and ATP but does not exhibit auto-phosphorylating activity. 32P-orthophosphate is also incorporated into the phosphoprotein in vivo.

Immunological identity of a 60 KD oncofetal protein induced in rats by chemical carcinogens and released by transformed cells

A 60,000 dalton (60 kd) oncofetal protein was previously shown to be produced by tumors in tumor-bearing rats and by target tissues within 3 weeks of carcinogen treatment. The factor is released to and accumulates in the blood in vivo and in the conditioned medium of cultured transformed cells in vitro. A polyclonal antibody produced against the 60 kd factor purified from the plasma of a rat carrying the N-2-fluorenylphthalamic acid-induced transplantable Hepatoma 7777, was tested against the 60 kd factor from various sources. Based on the results of immunoprecipitation of biochemical activity associated with the 60 kd factor, it was determined that these anti-60 kd antibodies cross-reacted with the factor released by a dimethylbenzanthracene-induced rat mammary carcinoma, with the factor in rat tumor cytosol and with rat spontaneous lymphoma cells, but not with a 60 kd factor isolated from pooled cancer patient plasma. Furthermore, these antibodies cross-reacted with the 60 kd factor induced within 21 days of treatment of the rats with a range of carcinogens from 8 chemical structural groups. The anti-60 kd factor antibodies did not cross-react with a 35 kd factor having similar biochemical activity found in normal adult cells.