Improved sensitivity for detection and quantitation of glycoproteins on polyacrylamide gels

Summary A simple and sensitive technique for staining glycoproteins on polyacrylamide gels utilizing periodic acid-Schiff (PAS) reagent is described. The novelty of this technique is an additional incubation of the PAS stained gels in an ethanolic solution of metabisulfite. The treatment results in an about 20-fold increase in the intensitiy of bands and the color is stable for at least 1 week at room temperature. Furthermore, the integrated optical density of the bands is proportional to the amount of glycoproteins. Amounts as small as 0.2 g of fetuin can be conveniently determined. As compared to other PAS staining methods, this procedure offers a remarkably improved sensitivity for detection and quantitation of glycoproteins following their electrophoretic separation on polyacrylamide gels.Author for correspondence and reprint requests: G.K., Department of Neurobiology and Anatomy, The University of Texas Medical School, P.O. Box 20708, Houston, Texas 77025, USA.     

Novel rearrangement of purines

Summary 6-Trichloromethyl-9-methylpurine (1) rearranges to 6-dichloromethyl-9-methyl-8-oxopurine (2) in aqueous mild acidic solution. The rearrangement is rationalized in terms of a reaction involving protonation, covalent hydration, prototropic equilibrium and/or a hydride transfer. An alternative mechanism involving a positive halogen compound and hypochlorous acid as an intermediary is also proposed. Compound1 condenses with 4,5-diaminopyrimidine to give the purine-pyrimidine Schiff base pair4.Acknowledgments. We are deeply indebted to Professor S. Cohen of the Sackler School of Medicine, Tel Aviv University (Ramat Aviv, Isreal) for his advice and encouragement. Support of this research by the Israel Cancer Association, the Ber-Lamsdorf Foundation Switzerland Israel and by the Advancement of Mankind Foundation, is gratefully acknowledged. We thank Proff. D. Arigoni and A. Eschenmoser, ETH Zürich, for their valuable proposals and comments on the mechanism of the rearrangement.     

Promotion of epithelial keratinization by N-methyl-N′-nitro-N-nitrosoguanidine in rat forestomach in organ culture

Summary The effect of N-methyl-N-nitro-N-nitrosoguanidine (MNNG) on epithelial differentiation of fetal rat forestomach was investigated in organ culture. When forestomach tissues removed from 16.5-day fetuses were treated with 5 g and 3 g of MNNG per ml for 1 h, epithelial keratinization was observed after 4 and 5 days, respectively, whereas it occurred after 6 days in control cultures. A clear dose-response relationship was found in the promotion of epithelial keratinization by MNNG.This work was supported by a Grant-in-Aid for Cancer Research from the Ministry of Education, Science and Culture, Japan.Acknowledgment. The authors wish to express their gratitude to Prof. T. Mizuno of the University of Tokyo for valuable suggestions.     

The biochemistry of ancient DNA in bone

The amount of DNA in ancient bone was determined by ethidium bromide staining after the removal of the potent Taq inhibitor, fulvic acid. A complete decalcification and a perfusion protocol were used to recover DNA from bone. A variety of purification techniques including molecular sieve, hydroxyapatite binding and Magic preparations yielded DNA that spanned from 3.4g/g of bone to below detectable limits. Fulvic acid was shown to interfere with the quantification of DNA derived from ancient human skeletal material one hundred to over seven thousand years old. Scanning UV in the 300 to 230 nm range is a simple and sensitive technique for documenting fulvic acid contamination in ancient bone extracts.     

Resistance to β-lactam antibiotics

-lactams have a long history in the treatment of infectious diseases, though their use has been and continues to be confounded by the development of resistance in target organisms. -lactamases, particularly in Gram-negative pathogens, are a major determinant of this resistance, although alterations in the -lactam targets, the penicillin-binding proteins (PBPs), are also important, especially in Gram-positive pathogens. Mechanisms for the efflux and/or exclusion of these agents also contribute, though often in conjunction these other two. Approaches for overcoming these resistance mechanisms include the development of novel -lactamase-stable -lactams, -lactamase inhibitors to be employed with existing -lactams, -lactam compounds that bind strongly to low-affinity PBPs and agents that potentiate the activity of existing -lactams against low-affinity PBP-producing organisms.Received 9 February 2004; received after revision 30 March 2004; accepted 19 April 2004     

Dissociation of glucocorticoid effects of C-21 steroids at high concentrations in thymocytes

Summary A dissociation between inhibition of RNA synthesis and cell lysis was observed when thymocytes of adrenalectomized rats were incubated with high concentrations of pregn-4-ene-11-ol-3, 20-dione and pregna-1,4-diene-11-ol-3,20-dione. In contrast, no dissociation of these effects was found with the typical glucocorticoids cortisol and corticosterone, nor with their 1,4-diene analogs under the same conditions.Acknowledgments. This work was supported by grants from the Consejo Nacional de Investigaciones Científicas y Técnicas de la República Argentina to the Instituto de Biologia y Medicina Experimental and the Programa de Regulación Hormonal y Metabólica as well as by financial help from the Secretaría de Ciencia y Tecnología.     

Conjugated terpenoid ketones: A new group of plant growth regulators

Summary ,-unsaturated terpenoid ketones have a root-inducing property on the hypocotyl cuttings ofPhaseoulus aureus. Significantly isopathcoulenone, (I) is distinctly more active in causeing rooting over IAA.Acknowledgment. The work was financially supported by the Punjab State Government, India, under the scheme Chemistry of some natural products and their significance in agriculture. The authors thank Dragoco, Holzminden, Federal Republic of Germany, for supplying the samples of monoterpenes.     

Thyroid function parameters during a selenium repletion/depletion study in phenylketonuric subjects

Phenylketonuric (PKU) subjects have a limited supply of selenium (Se) in their phenylalanine-restricted diet. A Se repletion (1 g Se/kg/day)/depletion study was conducted in PKU children to determine the effect of Se on thyroid function parameters.The initial plasma Se concentration (mean±SD: 0.26±0.12 mol/L, p<0.00003, n=10) and glutathione peroxidase (GSH-Px) activity (140±58 U/L, p<0.00003, n=10) were significantly lower compared to agematched controls. After 14 weeks of supplementation, the plasma Se concentration (mean ±SD: 0.74±0.20 mol/L) normalized (normal range: 0.57–1.15 mol/L, mean ±SD: 0.76±0.13 mol/L, n=32) and remained stable thereafter during repletion. Plasma GSH-Px activity reached normal values after 18 weeks of supplementation (312±57 U/L; normal range: 238–492 U/L, mean ±SD: 345±54 U/L, n=32) and increased significantly for up to eight weeks thereafter (332±52 U/L). Individual and mean thyroid parameters were initially normal in all cases. The mean concentrations of plasma thyroxine (T₄: p<0.025), free T₄ (FT₄: p<0.01) and reverse triiodothyronine (rT₃: p<0.005) decreased to 75% of their initial value within three weeks of Se supplementation and remained stable thereafter, within a normal physiological range during selenium supplementation. They increased back to their initial values three weeks (T₄: p<0.05, FT₄: p<0.05) and six weeks (rT₃: p<0.025) respectively, after the end of the supplementation. In conclusion, Se supplementation modifies thyroid function parameters in Se-deficient PKU subjects most likely by an increase in activity of type I 5-deiodinase (5-DIase I).Preliminary results of this study were presented as posters on the 4th International Congress on Trace Elements in Medicine and Biology, Chamonix, France (1993) and the 4th Joint Meeting of the Lawson Wilkens Pediatric Endocrine Society and the European Society for Paediatric Endocrinology, San Francisco, California, Ped Res (1993) 33: S93 (abstract 537). Support in part by Research Grants of Nutricia BV, (The Netherlands).     

The effect of dietary fibre on85Sr and47Ca absorption in infant rats

Summary The retention of bone-seeking radioisotopes⁸⁵Sr and⁴⁷Ca was about 20% lower in the femur of 6-day-old infant rats artificially fed on milk, milk supplemented with calcium and phosphorus, and milk supplemented with strontium when 6% cellulose type dietary fibre was added to the diet.Acknowledgments. This work was supported by a research grant from the Scientific Research Council of the Socialist Republic of Croatia. The authors wish to thank Mrs M. Buben, E. Herak, and K. Pribi for their technical assistance.     

Physiological importance of ω-3/ω-6 polyunsaturated fatty acids in man. An overview of still unresolved and controversial questions

Summary The essentiality of (-6) and (-3) fatty acids in mammals is well known. Nevertheless, some important points remain unclear concerning their implication in physiology. After a short discussion about the definition of essential fatty acids deficiency, this brief overview deals with some of these points, pointing out some of the unresolved questions. Different subjects are approached concerning the (-6) and (-3) fatty acids metabolism: desaturases, eicosanoids, production, as well as some of their metabolic effects on cell membranes, intestinal function, glucose and lipid metabolism, haemorheology.     

Genotype-specific reduction in methyl nitrosourea (MNU) induced sister chromatid exchanges (SCE) in vivo during aging

Summary We studied mice from five strains (BALB/c, C3H/HeSnJ, C57BL/6J, Cs^b and 129/ReJ) at two ages (young, 10±1 weeks; and old, 67±3 weeks) for the induction of sister chromatid exchanges (SCEs) in vivo by methyl nitrosourea (MNU). The SCE frequency is genotype-specific. The F₁ phenotype resembles the low responding parent. SCE induction is significantly lower in the older animals of each strain than their younger counterparts, and the reduction of SCE/cell with old age is strain-specific. A general explanation for these results must include strain differences in relative mutagenic sensitivity, genotype-specific pattern of reduction in DNA repair and other such factors affecting SCE formation, with old age.     

A rotating bottle culture method with continuous replacement of the gas phase

Summary A rotator culture method is described which provides a continuous flow of oxygenating gas to cultures in rotating bottles. The system maintains constant O₂ and CO₂ levels in the culture medium throughout the incubation period. It also provides a more stable pH than systems with sealed culture bottles.Acknowledgments. We would like to thank Mr B. Secker, Mr C. Hellon and Mr S. J. Ellis, of the Physiological Laboratory workshop, for constructing the rotator apparatus. We also thank the Medical Research Council, London, for financial support for the project.     

α-Adrenoceptor blocking action of hymenin,a novel marine alkaloid

Summary In the rabbit isolated aorta, hymenin (10^–6 M) a novel marine alkaloid, caused a parallel rightward shift of the dose-response curve for norepinephrine without affecting that for histamine or KCl, suggesting that hymenin is a competitive antagonist of -adrenoceptors in vascular smooth muscles.Acknowledgments. We thank Ms A. Kajiwara and Ms M. Hamashima of this institute for their technical assistance, and Prof. Y. Hirata of Meijo University for his encouragement.     

Purification of 20α-hydroxysteroid dehydrogenase from human erythrocytes

Summary 20-hydroxysteroid dehydrogenase was found in the supernatant after hemolysis of human erythrocytes, and the enzyme was isolated from the membrane-free hemolysate on a DEAE-cellulose column. The NADPH-generating system was essential for the reaction.Acknowledgments. We wish to thank Dr A. Tomoda, Department of Biochemistry, Kanazawa University School of Medicine, and Dr T. Yubisui, Department of Biochemistry, Medical College of Oita, for their advice and useful discussions.     

Inhibition of the growth of cariogenic bacteria in vitro by plant falvanones

Phytoalexins, defensive compounds produced by plants against microbial infections, were purified fromSophora exigua (Leguminosae) and their growth inhibitory effects on oral cariogenic bacteria were determined in vitro. Among three isolated compounds, 5,7,2,4-tetrahydroxy-8-lavandulylflavanone completely inhibited the growth of oral bacteria including primary cariogenic mutans streptococci, other oral streptococci, actinomycetes, and lactobacilli, at concentrations of 1.56 to 6.25 g/ml.     

Inhibitory effect of fasting on the glucagon-induced increase of liver phosphorylase A activity in rats

Summary The increase of liver phosphorylase A activity observed 1, 2 and 3 min after i.v. administration of 0.1 g kg^–1 glucagon to fed rats was found to be completely absent in 24 h fasted animals, although there is even an exaggerated liver cAMP response to glucagon after fasting.Acknowledgments. We would like to thank Mrs M. Fritzová and Mr F. Kovai for expert technical assistance.     

Blood plasma investigations by resonance raman spectroscopy: Detection of carotenoid pigments

Summary Using Raman spectroscopy, we demonstrate that low levels of -carotene, lycopene, and xanthophyll give rise to resonance enhanced bands in blood plasma. These results explain the significance of previously unidentified spectral maxima which have been related to the state of health of the blood donor.     

Zur Methodik der Gefrierschnitt-Mikroautoradiographie

Summary A microautoradiographic technique for frozen sections is described. The method makes it possible to follow the distribution of inhaled radon in the organism with high degree of accuracy. After inhaling radon, the mice were killed and stored in liquid air (freezing method ofRajewsky ⁹). The organ sections (5µ thick) were prepared by means of a freezing microtome in the Kryostat. A thin fixing and protecting layer of chromic alum gelatine was placed between the tissue-sections and the photographic emulsion. The exposure took place in an atmosphere of nitrogen.     

Neue Substitutionsreaktionen bei Steroiden

Summary The intramolecular substitution of the angular methyl groups in steroids by attack of O-radicals derived from 6-, 11, and 20-alcohols is discussed. Irrespective of their genesis, these radicals abstract a hydrogen atom from one of the said methyl groups, suitably oriented in-position. The C-radicals thus formed give different products depending on the neighbouring groups.It is shown that 20-hydroxy-18-C-radicals combine with iodine atoms, to give 18-iodides which can befurther substituted through radicals to give potential 18-oxo-compounds in high yield. In the case of 20-cyanohydrines, the intramolecular reaction of the 18-C-radical with the cyano groups gives rise to the new 18-cyano-20-oxo-steroids.With 6- and 11-O-radicalssingle substitution in 19-position predominates, since the steric arrangement of the primary product favours the intramolecular ether formation over a second substitution reaction.A general reaction scheme for these transformations is proposed.

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Vorgetragen vonCh. Meystre am 23. 9. 61 anlässlich der Sommerversammlung der Schweiz. Chem. Gesellschaft in Biel (vgl. Chimia15, (1961), im Druck).

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Über Steroide 180. Mitteilung. 179. Mitt. vgl.A. Wettstein, Exper.17, 329 (1961).