Detection of human papillomavirus in cervical smears. A comparison of in situ hybridization, immunocytochemistry and cytopathology

A comparison of different methods for the detection of cervical human papillomavirus (HPV) infection was made on patients attending the cervical dysplasia clinic. Cytomorphology, immunocytochemistry and in situ hybridization were compared for their ability to detect HPV. Separate cervicovaginal smears from 50 patients were tested for HPV types 6/11, 16 and 18 by in situ hybridization using 35S-labeled DNA probes. Duplicate smears from the same patients were Papanicolaou stained and evaluated for evidence of condylomatous and dysplastic changes. Twenty-five matching cervical biopsies were immunostained for HPV capsid antigen and tested by in situ hybridization for HPV DNA. The cytologic smears of 20 patients (40%) were positive for HPV DNA. Six patients had HPV 6/11, ten had HPV 16, three had HPV 18, and one had both HPV 6/11 and HPV 16. There was a high correlation between condylomatous cytopathology and antigen and DNA detection. One-third of the specimens with condylomatous changes were DNA negative by the tested probes, suggesting the presence of other HPV types in the genital tract.     

Solution conformation of the branch points of N-linked glycans: synthetic model compounds for tri'-antennary and tetraantennary glycans

The solution conformation of model compounds for the tri'-antennary and tetraantennary (six-arm) branch point of N-linked glycans has been determined through the use of chemical shift, relaxation, and nuclear Overhauser enhancement data. The object was to establish the conformation about the glycosidic linkages in the N-linked substructure GlcNAc(beta 1,6) [GlcNAc(beta 1,2)] Man(alpha)- by estimation of values for the appropriate glycosidic torsional angles. The GlcNAc(beta 1,6) linkage in a trisaccharide model compound was found to be constrained to a narrow rotameric subpopulation about the substituted Man C5-C6 bond (omega = -60 degrees) and a narrow range of possible phi - psi values. Free rotation about the Man C5-C6 bond was obstructed by unfavorable steric interactions between the GlcNAc(beta 1,6) and GlcNAc(beta 1,2) residues. A phi, psi value of 55 degrees, 190 degrees was found to be consistent with the NMR data for the GlcNAc(beta 1,6) linkage. However, the value of psi appears to be "virtual" in that the molecule is in equilibrium between two different values (90 degrees and 252 degrees). For the GlcNAc(beta 1,2) linkage, complete agreement between all the observed NMR parameters and all the calculated ensemble average values could only be obtained with a set of potential energy functions which included hydrogen bonding. Other choices of potentials yielded calculated values that disagreed with at least two of the observed quantities. As a result, we infer that an interresidue hydrogen bond is formed, and we find it to be between the GlcNAc(beta 1,2) ring oxygen and the Man C3 hydroxyl.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modification of galactose and N-acetylgalactosamine residues by oxidation of C-6 hydroxyls to the aldehydes followed by reductive amination: model systems and antifreeze glycoproteins

Amino acids and peptides have been attached to the C-6 hydroxyls of the galactose and the N-acetylgalactosamine by first oxidizing the C-6 hydroxyls to the aldehydes by galactose oxidase in the presence of small amounts of catalase, followed by reductive amination (-amino group) in the presence of cyanoborohydride. The activity of oxidized antifreeze glycoprotein was >70% of the original, and considerable activity has been retained with some substitutions on reductive amination using cyanoborohydride. The following were some activities retained (as compared with the oxidized antifreeze glycoprotein): Gly, 64; (Gly)₂, 88; (Gly)₃, 82; (Gly)₄, 70; Gly-Gly-NH₂, 44, Gly-Glu, 13; Gly-Leu, 40; Gly-Tyr, 57; Gly-Gly-Leu, 50; Gly-Gly-Phe, 30; and Gly-Gly-Val, 35. On amino acid analysis of acid hydrolysates, some release of the amino acid attached by amination occurred; e.g., Gly-Tyr gave 0.26 Gly and 0.49 Tyr per disaccharide.     

The effect of oxidants on biomembranes and cellular metabolism

During the reductive process in the tissues, the aerobes generate a number of oxidants. Unless these oxidants are reduced, oxidative damage and cell death would occur. Oxidation of plasma membrane lipids leads to autocatalytic chain reactions which eventually alter the permeability of the cell. The role of oxidative damage in the pathophysiology of diabetic complications and ischemic reperfusion injury of myocardium, especially the changes in the channel activity which may lead to arrhythmia have been studied. Hyperglycemia activates aldose reductase which could efficiently reduce glucose to sorbitol in the presence of NADPH. Since NADPH is also aldose required by glutathione reductase for reducing oxidants, its diversion would lead to membrane lipid oxidation and permeability changes which are probably responsible for diabetic complications such as cataractogenesis, retinopathy, neuropathy etc. Antioxidants such as butylated hydroxy toluene (BHT) and also reductase inhibitors prevent or delay some of these complications. By using patch-clamp technique in isolated frog myocytes, we have shown that hydroxy radicals generated by ferrous sulfate and ascorbate as well as lipid peroxides such as t-butyl hydroperoxide facilitate the entry of Na⁺ by oxidizing Na⁺-channels. Increased intracellular Na⁺ leads to an increase in Na⁺/Ca²⁺ exchange. The increased Na⁺ concentration by itself may produce electrical disturbance which would result in arrhythmia. Increased Ca²⁺ may affect proteases and may help in the conversion of xanthine dehydrogenase to xanthine oxidase, consequently increased production of super oxide radicals. Increased membrane lipid peroxidation and other oxygen free-radical associated membrane damage in myocytes has been demonstrated.     

Substrate reduction properties of dinitrogenase activated in vitro are dependent upon the presence of homocitrate or its analogues during iron-molybdenum cofactor synthesis

(R)-2-Hydroxy-1,2,4-butanetricarboxylic acid [(R)-homocitrate] has been has been recently reported to be an integral constituent of the otherwise thought to be inorganic iron-molybdenum cofactor of dinitrogenase [Hoover, T.R., Imperial, J., Ludden, P.W., & Shah, V.K. (1989) Biochemistry 28,2768-2771]. Different organic acids can substitute for homocitrate in an in vitro system for iron-molybdenum cofactor synthesis and incorporation into dinitrogenase [Hoover, T.R., Imperial, J., Ludden, P.W., & Shah, V. K. (1988) Biochemistry 27, 3647-3652]. Dinitrogenase activated with homocitrate-FeMo-co was able to reduce dinitrogen, acetylene, and protons efficiently. Homoisocitrate and isocitrate dinitrogenases did not reduce dinitrogen or acetylene, but showed very high proton reduction activities. Citrate and citramalate dinitrogenases had very low dinitrogen reduction activities and intermediate acetylene and proton reduction activities. CO inhibited proton reduction in both these cases but not in the case of dinitrogenases activated with other homocitrate analogues. By use of these and other commercially available homocitrate analogues in the in vitro system, the structural features of the homocitrate molecule absolutely required for the synthesis of a catalytically competent iron-molybdenum cofactor were determined to be the hydroxyl group, the 1- and 2-carboxyl groups, and the R configuration of the chiral center. The stringency of the structural requirements was dependent on the nitrogenase substrate used for the assay, with dinitrogen having the most stringent requirements followed by acetylene and protons.     

Glucose utilization and growth response to protein hydrolysates byFusobacterium species

Glucose uptake was measured in the supernatants of 18 strains ofFusobacterium species cultured in BM medium. Some species, such asF. nucleatum andF. necrophorum, used between 25% and 48% of the glucose in the medium, but the terminal pH remained near neutral. By contrast, strains ofF. mortiferum andF. necrogenes used on average over 90% of the available glucose in the medium and produced a predictably low acidic pH. Strains ofF. varium used between 86% and 91% of the glucose present but produced a near neutral pH of between 5.8 and 5.9. The metabolic fate of glucose inF. varium was, therefore, examined in more detail. Glucose stimulated the growth of this species, and [¹⁴C]glucose was incorporated into the metabolic end products and various cellular components. Protein hydrolysates, tested for their growth-promoting effects onFusobacterium species, produced two general growth response patterns. Most species grew prolifically on trypticase, proteose peptone, and yeast extract, but poorly in casamino acids and vitamin-free casamino acids. Growth in bactocasitone was poor, but for three species,F. necrophorum, F. varium, andF. nucleatum, there was an approximately linear growth response up to 0.5%. These results suggest a major role for nitrogen metabolism but do not preclude glucose as an energy source in at least some species ofFusobacterium.     

Effect of alkylated and intercalated DNA on the generation of superoxide anion by riboflavin

Superoxide anion (O ₂ ^.– ) was photogenerated upon illumination of riboflavin in fluorescent light. The rate of O ₂ ^.– formation was stimulated by double stranded DNA but not by denatured DNA or RNA. Depurinated DNA, which was predominantly depleted in guanine residues, did not exhibit the stimulatory effect, indicating an interaction of riboflavin, or active oxygen species derived from it, with guanine bases. Also, the stimulation of O ₂ ^.– photogeneration was not observed with ethidium bromide but was seen with proflavin-intercalated DNA. Since ethidium bromide intercalates preferentially between purines and pyrimidines, and proflavin prefers dA-dT rich sites, these results were interpreted to suggest that the interaction of riboflavin with DNA is mainly with GC or CG base pairs.     

Characteristics of glutamate dehydrogenase, a new diagnostic marker for the genus Fusobacterium

Enzymes representative of carbohydrate and nitrogen metabolism were screened for their presence and activity amongst species of the genus Fusobacterium. Glutamate dehydrogenase (GDH) was reliably detected in all 25 strains studied. The pH profile of this enzyme and the DNA base composition of selected strains were also determined. DNA base composition of selected strains ranged between 28-32.9 mol% G + C. GDH was active between pH 7.5-11.5 but two pH profiles of activity, with optima at 9.5 and 10.5, were discernible among species. Apart from Fusobacterium nucleatum, which had a heterogeneous enzyme pattern, the GDH electrophoretic mobility was constant within a species but in a few cases the enzyme bands overlapped. A combination of the pH profile, the GDH electrophoretic pattern and the DNA base composition provided clear separation of the test organisms into discrete groups; however, a larger number of strains must be examined before the full potential of these tests can be evaluated.     

Macrophylloside, a flavone glucoside from Primula macrophylla

A new flavone glucoside macrophylloside has been isolated from the whole plant of Primula macrophylla and its structure was determined by spectroscopic methods as 2′-hydroxy-7-O-β- -glucopyranosyloxyflavone. Sitosterol glucoside was also isolated for the first time from this plant.