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.     

A quantitative method for the detection and localization of quantum-limited events from radionuclides in cells and tissue sections by computer-enhanced video microscopy

Cellular dynamics often involve extremely low concentrations of biologically active substances, which can be radiolabeled and detected, localized and quantitated by autoradiography. The latter may require exposures from a few days to many months. The objective of this research was to demonstrate the feasibility of reducing this long period of data collection by one to two orders of magnitude, while maintaining or improving the spatial resolution and localization in tissues and the quantitative characteristics inherent in autoradiography. A mathematical model describing the complete system was generated using energy partition calculations to estimate photon production via scintillant per H3 beta particle emission and to estimate the subsequent photon capture based upon imaging system parameters and microscope geometry. Calculations showed that, typically, a single tritium beta particle produces a maximum of 5.8 X 10(3) photons. A photon-limited camera and microscope imaging system were selected and optimized in conjunction with a specially developed physical scintillation model. Results showed that the number of detected photoevents increases monotonically with both signal integration time and, independently, with the concentration of the radionuclide. Consequently, this work demonstrates that video microscopy imaging methods can spatially and temporally quantify very low concentrations of radiolabeled substances and can reduce data acquisition times.     

Functional drug targeting to erythrocytes in vivo using antibody bearing liposomes as drug vehicles

Covalent attachment of anti-erythrocyte F(ab')2 to the liposome surface has recently been shown to considerably enhance the liposome binding to erythrocytes in vivo. These antibody bearing liposomes have now been found quite effective as vehicles for delivering the antimalarial drug, chloroquine, to erythrocytes in Plasmodium berghei-infected mice. This demonstrates the usefulness of antibody targeted liposomes as carriers for site-specific drug delivery.     

The alternating conformation of analogues of poly[d(AT)].

Synthetic DNAs were prepared containing 6-methyl adenine (m6A) in place of adenine and 5-ethyl uracil (Et5U) or 5-methoxymethyl uracil (Mm5U) in place of thymine. All three modifications destabilized duplex DNAs to varying degrees. The binding of ethidium was studied to analogues of poly[d(AT)]. There was no evidence of cooperative binding and the "neighbour exclusion rule" was obeyed in all cases although the binding constant to poly[d(m6AT)] was approximately 6 fold higher than to poly[d(AT)]. 31P NMR spectra were recorded in increasing concentrations of CsF. Poly[d(AEt5U)] showed two well-resolved signals separated by 0.55 ppm in 1 M CsF compared to 0.32 ppm for poly[d(AT)] under identical conditions. In contrast, poly[d(AMm5U)] and poly[d(m6AT)] showed two signals separated by 0.28 ppm and 0.15 ppm respectively, only when the concentration of CsF was raised to 2 M. The signals for poly[d(AT)] in 2 M CsF were better resolved and were separated by 0.41 ppm. These results suggest that minor modifications to the bases may have conformational effects which could be recognized by DNA-binding proteins.     

Effect of DDT on hepatic mixed-function oxidase activity in normal and vitamin A-deficient rats

Feeding of vitamin A-deficient diet to male weanling rats for 10 weeks resulted in significant decrease in the body weight and marked reduction in the hepatic vitamin A content. The levels of hepatic phase I microsomal enzymes cytochrome P-450, cytochrome b5, aminopyrine N-demethylase and arylhydrocarbon hydroxylase were found to be substantially reduced by vitamin A-deficiency. Also, the activity of phase II microsomal UDP - glucuronyl transferase enzyme was significantly decreased in deficient animals. Following repeated oral administration of DDT (15 mg/kg/body wt/day) for 21 days, the phase I microsomal enzymes were induced to a greater extent in controls as compared to deficient animals. UDP - glucuronyltransferase remained insensitive to DDT induction. The results imply that the capacity for induction of the hepatic mixed-function oxidase enzyme system is impaired in deficient animals concurrently exposed to DDT.     

A simple and sensitive spectrophotometric method for the quantitative determination of solid supported amino groups

A simple and sensitive method for the quantitative determination of free amino groups on solid support is described. This approach is a modification of Ngo's [(1986) J. Biochem. Biophys. Methods 12, 349-354] method reported earlier. The method is based on the reaction of the solid support with an excess of 5'-O-(4,4'-dimethoxytrityl)-thymidine-3'-O-(2,4-dinitrophenyl) succinate (DTDS) in the presence of a catalytic amount of 4-dimethylaminopyridine. After removing the excess reagent, solid support is treated with perchloric acid to release 4,4'-dimethoxytrityl cation into the solution. The released 4,4'-dimethoxytrityl cation, which has a strong absorption at 498 nm (epsilon 498 = 70,000), is then determined spectrophotometrically. A comparative study of DTDS, N-succinimidyl-3-(2-pyridyldithio)propionate and 4,4-dimethoxytrityl chloride is also included. The method was found to be very useful to determine those amino groups which are available for functionalization of solid supports, especially, monitoring the functionalization of solid supports for affinity chromatography and synthesis of biopolymers.     

A novel primase-free form of murine DNA polymerase alpha induced by infection with minute virus of mice

Two species of DNA polymerase alpha free of primase activity were identified in extracts of Ehrlich mouse cells that had been infected with minute virus of mice. Primase-free forms of DNA polymerase alpha eluted with 150 and 180 mM NaCl during ion-exchange chromatography on DEAE-cellulose columns, exhibited sedimentation coefficients of 11 S and 8.2 S, respectively, and were inhibited by aphidicolin, N2-(p-n-butylphenyl)-9-(2-deoxy-beta-D-ribofuranosyl)guanine 5'-triphosphate, and 2-(p-n-butylanilino)-9-(2-deoxy-beta-D-ribofuranosyl)adenine 5'-triphosphate. The ratio of primase-free DNA polymerase alpha to the DNA polymerase alpha-primase complex increased from 1.5 to greater than 100 during the course of infection, and free primase was produced during the MVM replicative cycle.     

Role of endogenous opioids and histamine in morphine induced emesis

The role of opioid and histaminergic system in morphine induced emesis was investigated in dogs. Morphine (25 micrograms, icv) consistently evoked emesis with an average latency of 195 +/- 29 sec which was fully accounted for by an action on the chemoreceptor trigger zone (CTZ) as its ablation rendered animals refractory to vomiting. Intraventricular pretreatment with opioid antagonist naloxone, histamine H1 antagonist mepyramine and H2 antagonists metiamide and cimetidine afforded protection to icv morphine emesis. The CSF histamine concentration was significantly raised 5 min after icv morphine administration. The results suggest that both endogenous opioid and histamine are involved in morphine emesis. Naloxone in high doses (1600 micrograms, icv) elicited emesis which was not blocked by CTZ ablation confirming our earlier report.