The cost-effectiveness of treating women with a cervical vaginal smear diagnosis of atypical squamous cells of undetermined significance.

OBJECTIVE: The purpose was to determine the optimal treatment protocol for women with a cervical vaginal diagnosis of atypical squamous cells of undetermined significance. STUDY DESIGN: By means of decision analysis, 8 strategies were compared in terms of cost, life expectancy, cost-effectiveness, and the number of cancers and complications from treatment. Data were obtained from the medical literature and the University of Iowa Hospitals and Clinics. RESULTS: Compared with more aggressive strategies, such as those that use immediate colposcopy, strategies featuring repeated smears were less expensive and carried fewer complications but had lower life expectancies per patient and more cancers. The strategy of repeating a smear annually had a lower cost per patient than did the other strategies, ranging from $112 to $989, and had a similar discounted life expectancy to that of the strategy with the longest discounted life expectancy. CONCLUSIONS: In most clinical scenarios strategies that used repeated smears were the most cost-effective.

Problems encountered with the Bethesda System: the University of Iowa experience.

This paper provides a descriptive summary of 1 year's experience with the Bethesda Cytologic Classification System at the University of Iowa Hospitals and Clinics. Cytotechnologists and pathologists criticized the failure of the new classification system to provide microscopic criteria for the following categories: adequacy of the smear (satisfactory, less than optimal, unsatisfactory), atypical squamous cells of undetermined significance, atypical glandular cells of undetermined significance, and inflammation-associated changes. Clinicians criticized the "less than optimal" category and the reporting of koilocytosis, moderate dysplasia, and endometrial cells. Suggestions for modifying the Bethesda System include: eliminating the less than optimal response, defining what constitutes an unsatisfactory smear, eliminating the terms low-grade and high-grade squamous intraepithelial lesion, and changing the reporting of endometrial cells.

The second-messenger system for peptide hormones.

There are three major hormone classes--peptide, steroid, and the newly defined growth factors--each with its own system for signal transduction in the cell. Two interdependent theses are proposed for the peptide hormone: that incoming signal transduction requires coupling to a G protein in a second-messenger pathway, and that second-messenger redundancy assures checks and balances in cell regulation.

Immunolocalization of ornithine decarboxylase in rat heart atria.

Mammalian atrial myocytes secrete a potent natriuretic and diuretic factor, atrial natriuretic peptide (ANP), in response to volume expansion or elevations of right atrial pressure. ANP is synthesized in the atrial atrial myocytes and stored in cytoplasmic secretory granules. Ornithine decarboxylase (ODC) is the initial rate-limiting enzyme in the biosynthesis of polyamines, organic cations which are essential for various aspects of DNA, RNA and protein synthesis, structure, and function. The enzyme has been reported to be localized predominantly in the cytoplasm. A polyclonal antibody elicited against ODC was used to analyse the intracellular distribution of the enzyme protein within atrial myocytes at the ultrastructural level through the use of a post-embedding immunogold technique. In control animals, the density of gold particles associated with the atrial granules was seven to 30-fold higher than that detected in association with other subcellular structures. Administration of Isoproterenol increased atrial of Isoproterenol increased atrial ODC activity 18-fold and the density of the immunolabelling of the atrial myocytes five-fold. Statistically significant increases in the density of labelling after stimulation occurred in association with the atrial granules and the nucleus. After isoproterenol stimulation, 60% of the immunodetectable ODC protein in the atrial myocyte was found in association with the atrial granules. The atrial granules were demonstrated to contain ANP by immunocytochemical analysis of adjacent sections.

Species specificity of antibodies to regulatory subunits of cyclic AMP-dependent protein kinases.

Polyclonal antibodies were generated against regulatory subunits (RI and RII) of type-I and type-II cAMP-dependent protein kinases from rat skeletal muscle. Western immunoblot analyses showed specific cross-reactivity of rat and bovine RI with anti-RI. Similarly, RII from both species was specifically recognized by anti-RII. Quantitative immunoassays, using antisera against proteins from either species, indicated selectivity towards regulatory subunits from the same species. Molecular basis for this selectivity was examined by comparison of peptide maps of 32P-8-azido-cAMP-labelled or autophosphorylated peptides. Detailed analysis of two-dimensional peptide fingerprints demonstrated extensive homology between either RI or RII from the two species. The data suggests that the overall protein-chemical and functional determinants characterizing type-I and type-II regulatory subunits of cyclic AMP dependent protein kinase from different species are substantially similar. However, minor differences in structure, also predicted by amino-acid sequences for RI and RII obtained by molecular cloning, may account for the distinct immunological properties of the proteins from rat and bovine tissues.

Increase in liver protein phosphatase-1 in spontaneously diabetic Chinese hamsters.

Two broad-specifically protein phosphatases, termed protein phosphatase-1 (PrP-1) and protein phosphatase-2A (PrP-2A), accounting for all the hepatic activity regulating glycogen phosphorylase, were measured in spontaneously diabetic Chinese hamsters exhibiting persistent glycosuria. When compared with genetically related inbred sublines free of glycosuria, diabetic animals demonstrated approximately 25% increase in PrP-1 activity measured either in crude tissue extracts or in cytosols fractionated by ion-exchange chromatography. No significant alteration in total PrP-2A activity was observed in the diabetic animals. These findings indicate that a specific change in hepatic PrP-1 is associated with genetically acquired diabetes in Chinese hamsters. In contrast to reported data using animals with experimentally induced diabetes mellitus, hepatic PrP-1 was increased in the spontaneously diabetic Chinese hamsters. The data suggests that distinct alterations in PrP-1 and associated metabolic consequences are exhibited by different types of diabetes.

A new technique for removal of amorphous phase tissue water without ice crystal damage: a preparative method for ultrastructural analysis and immunoelectron microscopy.

An apparatus has been produced that can remove amorphous phase tissue water via molecular distillation without devitrification or rehydration. This method represents a fundamental advance in tissue preparation, making possible for the first time ultrastructural localization of soluble molecular entities without the problems of alteration, re-distribution, and loss which have plagued conventional techniques. Fresh slices of rat brain, liver, or kidney, and monkey retinal tissue were cryofixed by bounce-free, metal mirror cooling on copper bars immersed in liquid nitrogen (LN2). Tissue transferred under LN2 was then placed in a precooled copper specimen block, which was subsequently lowered into a LN2-cooled stainless steel chamber. After rough pumping at 1 X 10(-3) mbar with a mechanical pump to remove LN2, the chamber was evacuated with a cryopump or turbomolecular pump to achieve a hydrocarbon-free, ultra-high vacuum of 1 X 10(-8) mbar. Equilibrium temperature in the chamber before the drying cycle was -192 degrees C. The copper specimen block was equipped with a thermocouple and a programmable feedback-controlled heating circuit. Tissue was dried by increasing the specimen block temperature 1 degree C/hr during the critical drying phase while monitoring the rate of water removal with a partial pressure analyzer. Results obtained indicate that drying is complete below the devitrification temperature of amorphous phase tissue water. Dried tissue was fixed with osmium tetroxide vapor, vacuum-embedded in a low-viscosity epoxy resin, sectioned, stained, and viewed with the electron microscope. Processed tissue exhibits excellent morphological preservation without the use of pre-fixation or cryoprotective agents. Thin sections of this tissue are excellent for immunocytochemical staining and electron microprobe analysis.

Purification of cyclic AMP- and cyclic GMP-dependent protein kinases from rat skeletal muscle.

Cyclic AMP-dependent protein kinase (cAMP-PrK) regulatory subunits, RI and RII, and cyclic GMP-dependent protein kinase (cGMP-PrK) have been simultaneously purified from skeletal muscle, utilizing sequential affinity chromatography on cyclic AMP-Sepharose. Rat skeletal muscle extract was chromatographed over DEAE-cellulose. Appropriate fractions, enriched in RI, RII or cGMP-PrK were further purified by affinity chromatography on cAMP-Sepharose. The protein kinase units were specifically eluted with cAMP or cGMP. A novel procedure, using two affinity columns, differing in their linkage of cAMP via either N6 or C-8 bonds, was developed to obtain RII free of other cyclic nucleotide binding proteins. In all cases, affinity chromatography was followed by HPLC gel exclusion chromatography to remove residual contaminating proteins. Proteins were purified to essential homogeneity as judged by silver stained SDS polyacrylamide gels. This procedure yields protein kinase subunits of high purity, and may be applicable to the isolation of these proteins from other sources.

Effect of retinoic acid on cellular content and human parathyroid hormone activation of cyclic adenosine 3':5'-monophosphate-dependent protein kinase isoenzymes in clonal rat osteogenic sarcoma cells.

Pretreatment with 10(-8) M retinoic acid for 4 days caused changes in three distinct components of the parathyroid hormone (PTH)-stimulated cyclic adenosine 3':5'-monophosphate response in a clonal rat osteogenic sarcoma cell line, UMR 106-06: the amplitude of the cyclic adenosine 3':5'-monophosphate response to PTH was moderately increased after pretreatment with retinoic acid; while the cellular content of the two isoenzymes of the cyclic adenosine 3':5'-monophosphate-dependent protein kinase was approximately equal in control cells, retinoic acid pretreatment was associated with a marked increase in the ratio of type II to type I holoenzyme activity. This change might be due to a decrease in the type I holoenzyme as suggested by immunofluorescence detection of decreased type I regulatory subunit in fixed cells together with the relative decrease in type I holoenzyme determined biochemically; there was a marked alteration of the pattern of PTH-stimulated protein kinase isoenzyme activation from predominantly type I isoenzyme in control cells to almost exclusively type II isoenzyme in retinoic acid-treated cells. Growth inhibition by submaximal amounts of PTH and retinoic acid when added together was greater than that for either agent alone.

Characterization of cyclic AMP-binding proteins in rat sertoli cells using a photoaffinity ligand.

Protein-bound cyclic AMP (cAMP) levels in cultured rat Sertoli cells have been determined after exposure to follicle-stimulating hormone (FSH) and agents which elevate intracellular cAMP or mimic cAMP action. Changes in the content of protein-bound cAMP were correlated with changes in receptor availability determined by measuring [3H] cAMP binding. Using the photoaffinity analog of cAMP, 8-N3 [32P] cAMP, two major cAMP-binding proteins in Sertoli cell cytosol, with molecular weights of 47 000 and 53 000 daltons, were identified as regulatory subunits of type I and type II cAMP-dependent protein kinases, respectively. Densitometric analysis of autoradiograms demonstrated differential activation of the two isozymes in response to treatment with FSH and other agents. Results of this study demonstrate the value of measuring changes in protein-bound cAMP and the utility of the photoaffinity labeling technique in correlating hormone-dependent processes in which activation of cAMP-dependent protein kinase occurs.

Immunocytochemical localization of cyclic GMP, cGMP-dependent protein kinase, calmodulin and calcineurin in Paramecium tetraurelia.

The localization of cGMP, cGMP-dependent protein kinase, calmodulin and the calmodulin-binding protein calcineurin in Paramecium tetrauelia cells has been examined with immunocytochemical methods. These molecules appeared to be localized to a large extent in the cilia of this protozoan. To ascertain that antibodies had access to all cellular compartments we have used three different preparations for immunocytochemistry: (i) with 'whole cell' preparations immunofluorescent staining for the four molecules was mainly visible in the cilia; (ii) in 'deciliated' Paramecium, staining for cGMP and calmodulin was found in regular patterns on the cell surface most likely representing kinetosomes; (iii) using 'sectioned cells', additional cytoplasmic calmodulin appeared to be associated with glycogen particles as evidenced by the disappearance of the granular staining pattern after preincubation with alpha-amylase. In contrast, cGMP, cGMP-dependent protein kinase and calcineurin fluorescence was only very weak and diffuse in cell bodies. No nuclear fluorescence was detectable after staining with any of the antibodies. Because of the colocalization of cGMP, cGMP-dependent protein kinase, a guanylate cyclase-calmodulin-complex, and calcineurin in cilia from Paramecium, an involvement of these components in the regulation of ciliary activity is discussed.

Methods of cervical priming.

It is now well recognized that a priming agent without untoward effects on mother or fetus is highly desirable when an indicated induction is required in the face of an unripe cervix. There appears to be a potential role for Laminaria, estrogens, relaxin, and various forms and routes of administration of prostaglandins because efficacy has been reported with all of these methods. With careful selection of patients and judicious fetal monitoring, there are multiple reasonable modes available, although an ideal priming agent has not yet emerged.

Follicle-stimulating hormone-dependent phosphorylation of vimentin in cultures of rat Sertoli cells.

Endogenous protein phosphorylation was investigated in cultured rat Sertoli cells after treatment with follicle-stimulating hormone (FSH) and pharmacological agents that activate cAMP-dependent protein kinases. In intact Sertoli cells, both phosphorylation and dephosphorylation of proteins occurred in response to treatment with these agents. Studies using cell-free preparations suggest that four phosphoproteins phosphorylated by cAMP or the catalytic subunit of cAMP-dependent protein kinase were also phosphorylated in a FSH-dependent manner in intact cells. These data suggest that FSH-dependent phosphorylation in Sertoli cells occurs through activation of a cAMP-dependent protein kinase. A FSH-dependent phosphoprotein with a molecular weight of 58,000 was identified as the intermediate filament protein vimentin, based on its migration in two-dimensional gels and its peptide map. The cellular distribution of vimentin was monitored by immunofluorescence in Sertoli cells after treatment with FSH. Results of this study support a role for intermediate filaments in FSH-dependent events in Sertoli cells.

Immunofluorescent localization of cGMP, cGMP-dependent protein kinase, calmodulin and cAMP in the rat uterus. Effects of estrogen treatment.

Cyclic guanosine 3',5' monophosphate (cGMP), cGMP-dependent protein kinase, calmodulin and cyclic adenosine 3',5' monophosphate (cAMP) were localized in the uterus of the immature rat by an indirect immunofluorescence technique. cGMP, cGMP-dependent protein kinase and calmodulin were detected predominantly along epithelial and myometrial plasma membranes and in the adjacent cytoplasm. In contrast, cAMP immunoreactive material was found principally in the cytoplasm of connective tissue. After administration of 17 beta-estradiol, similar time-dependent changes were observed in the localization of cGMP, cGMP-dependent protein kinase and calmodulin in all uterine cell types. For the three compounds, nucleolar-like distribution of the immunofluorescence appeared approximately 12 h after treatment. A more dispersed, reticular distribution of the nuclear fluorescent staining was observed 20-24 h after hormonal treatment. Estrogen did not affect the localization of cAMP. The simultaneous mobilization of cGMP, cGMP-dependent protein kinase and calmodulin towards the same nuclear loci suggests concerted roles for these three molecules in nuclear metabolic processes during the development of the uterotrophic action of estrogens.

Localization of the catalytic subunit of cyclic AMP-dependent. Protein kinase in cultured cells using a specific antibody.

We developed a specific antibody to the catalytic subunit (C-subunit) of cyclic AMP-dependent protein kinase and used it to localize C-subunit in cultured cells. C-subunit antigen was purified from bovine cardiac muscle and cross-linked to hemocyanin with glutaraldehyde. Immunized goat serum showed a low titer of antibody after boosting; it was enriched 100-fold by affinity chromatography on catalytic subunit-Sepharose. The antibody immunoprecipitated C-subunit from type I and type II holoenzyme and depleted enzymatic activity from solution. At 12.5 nM antigen, 1 microgram antibody immunoprecipitated 10 ng of C-subunit. Immunoprecipitation of 35S-labeled cell extracts and 125I-antibody detection on nitrocellulose paper revealed that the antibody specifically reacts with C-subunit in Chinese hamster ovary (CHO) whole cell extracts. Using indirect immunofluorescence to localize C-subunit, we found a pattern of diffuse staining in the cytoplasm of CHO cells with little or no nuclear staining. A similar distribution of the enzyme was observed in Swiss 3T3 cells, bovine endothelial tracheal cells, human lung fibroblasts and NRK cells. Treatment of CHO cells with 8-bromo-cyclic AMP produced no change in the pattern or intensity of immunofluorescence. We conclude that the majority of C-subunit is localized in cytoplasm and that in cultured fibroblasts exposure to cyclic AMP analogues causes no apparent redistribution of catalytic subunit.