Simplified radiographic-surgical template for placement of multiple, parallel implants.

A radiographic-surgical template can facilitate consultation with a surgeon and patient when implant-supported restorations are planned. A template that provides radiographic evaluation of the implant site and precise or modified surgical placement is presented.

Composite veneered acrylic resin provisional restorations for complete veneer crowns.

Restorations that are remade to improve esthetics, teeth with pronounced gingival-to-incisal color contrast, or high-value translucent teeth can limit the capability of shaded acrylic resin provisional restorations to satisfy the esthetic demands of certain patients. This article describes a procedure to incorporate a composite veneer in an acrylic resin provisional restoration. Improved optical properties of microfilled composite are combined with the excellent marginal seal and contour of acrylic resin. Precise control of color, translucency, and surface texture provide excellent interim esthetics and a better guide for the definitive prosthesis.

Identification of mammosomatotrophs in the turkey hen pituitary: increased abundance during hyperprolactinemia.

We have previously reported that the hyperprolactinemia in incubating turkey hens is associated with recruitment of lactotrophs in the pituitary gland. In this study we have used double immunofluorescence and in situ hybridization histochemistry to 1) identify mammosomatotrophs in the anterior pituitary gland of egg-laying turkey hens and incubating hens, and 2) verify PRL gene expression within mammosomatotrophs by colocalizing PRL messenger RNA in GH-immunoreactive (ir) cells. The pituitaries of laying and incubating turkey hens were collected, and the midsagittal sections were dual labeled for either PRL and GH or PRL messenger RNA and GH. The plasma PRL concentrations were higher in incubating hens (231 +/- 10.6 ng/ml) than in laying hens (43 +/- 7.4 ng/ml; P < 0.01). In the midsagittal pituitary sections, mammosomatotrophs were predominantly found scattered in the caudal lobe of the anterior pituitary gland, in the ventral half of the cephalic lobe, and at the junction of cephalic and caudal lobes. In incubating hens, the proportion of mammosomatotrophs was 7.4 +/- 1.52% (mean +/- SEM) of the total number of GH-ir and/or PRL-ir cells counted, which was significantly higher (P < 0.05) than that found in laying hens (0.6 +/- 0.23%). Furthermore, PRL gene expression was observed in many GH-ir cells in the incubating hen pituitary gland. These data suggest that 1) mammosomatotrophs are present in the turkey pituitary gland, and 2) there is an increased abundance of mammosomatotrophs in the incubating turkey hen that may contribute to hyperprolactinemia.

An improved system for packaging recombinant adeno-associated virus vectors capable of in vivo transduction.

Adeno-associated virus (AAV) vectors are potentially useful for gene therapy of a number of human diseases. However, the use of these vectors has been limited by the lack of stable vector-packaging cell lines. The difficulties in developing packaging cell lines relate to low levels of rep gene expression from the AAV-p5 promoter, and to the propensity of Rep proteins to suppress continued growth of immortalized cell lines. We describe here two new techniques which allow these problems to be circumvented. First, we have demonstrated that expression of rep from the human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter results in a 10-fold improvement of packaging efficiency. Second, we have overcome the inefficiency of vector plasmid transfection by generating cell populations containing rescuable AAV recombinant genomes. These improvements yielded a net increase of 50-fold in the packaging efficiency of the AAVp5neo and AAVp5lacZ recombinant vectors. The AAVp5lacZ vector packaged with this method was administered systemically to recently weaned C57BL mice, and mediated efficient expression of the beta-galactosidase reporter gene in cells of the airway epithelium and spleen. This indicates the in vivo activity of these vector stocks, and their potential utility for gene therapy.

Stable in vivo expression of the cystic fibrosis transmembrane conductance regulator with an adeno-associated virus vector.

Adeno-associated virus (AAV) vectors expressing the normal cystic fibrosis transmembrane conductance regulator (CFTR) cDNA complement the cystic fibrosis (CF) defect in vitro. Unlike other DNA virus vectors, AAV is a stably integrating virus, which could make possible long-term in vivo complementation of the CF defect in the airway epithelium. We report AAV-CFTR gene transfer and expression after infection of primary CF nasal polyp cells and after in vivo delivery of AAV-CFTR vector to one lobe of the rabbit lung through a fiberoptic bronchoscope. In the rabbit, vector DNA could be detected in the infected lobe up to 6 months after administration. A 26-amino acid polypeptide sequence unique to the recombinant AAV-CFTR protein was used to generate both oligonucleotide probes and a polyclonal antibody which allowed the unambiguous identification of vector RNA and CFTR protein expression. With these reagents, CFTR RNA and protein were detected in the airway epithelium of the infected lobe for up to 6 months after vector administration. AAV vectors do, therefore, efficiently promote in vivo gene transfer to the airway epithelium which is stable over several months. These findings indicate that AAV-CFTR vectors could potentially be very useful for gene therapy.

Expression of the cystic fibrosis transmembrane conductance regulator from a novel adeno-associated virus promoter.

Adeno-associated virus type 2 (AAV) vectors have been used for gene expression in respiratory epithelial cells and may be useful in gene therapy for diseases like cystic fibrosis (CF) which affect the airways. The AAV p5 promoter together with the AAV inverted terminal repeat (ITR) forms a 263-base pair cassette which mediated efficient expression in a CF bronchial epithelial cell line. We report here that the ITR itself can mediate gene expression. In stable transfection assays, AAV-CF vectors expressing the full-length cystic fibrosis transmembrane conductance regulator (CFTR) cDNA from either the p5 promoter or the ITR restored cAMP regulation of the chloride efflux characteristic of CFTR function. An AAV-ITR-CF vector deleted for the amino terminus of CFTR was also functional. This vector was packaged into AAV particles and used to transduce cells without selection. Transduced cells also exhibited cAMP-regulated Cl- efflux. The complemented cell lines showed increased levels of CFTR protein immunofluorescence, and the presence of intact AAV-CF vector sequence was confirmed by Southern blot analysis of rescued vector sequences. These studies provide novel insights into AAV gene expression, and this newly described promoter allows for the production of AAV vectors expressing CFTR in those differentiated cells affected in CF.

Gene expression from adeno-associated virus vectors in airway epithelial cells.

Lung diseases such as cystic fibrosis (CF) might be treated by gene therapy using viral vectors delivered to the airway. One potential vector is the defective human parvovirus, adeno-associated virus (AAV). We examined the AAV p5 transcription promoter for gene expression in immortalized cell lines derived from the airway (IB3-1) or pancreas (CFPAC-1) of CF patients. AAV vectors expressing the prokaryotic genes cat (pAAVp5cat) or neo (pAAVp5neo) from the p5 promoter were evaluated after introduction into IB3-1 or CFPAC-1 cells by lipofection. In transient assays in both cell lines, the cat gene was expressed 5- to 10-fold more efficiently from the p5 promoter than from a simian virus 40 early gene promoter (pSVcat). IB3-1 cells were transformed stably to geneticin resistance by pAAVp5neo at a 5-fold higher efficiency than by an SVneo vector. The AAV inverted terminal repeat (ITR) region immediately upstream of the p5 promoter appears to have an enhancer effect and the promoter also contains a CREB site which confers a response to forskolin. In IB3-1 cells, expression of the cat gene from a p5 promoter was decreased about 5-fold by deletion of both the upstream ITR and the CREB site. The AAVp5neo vector was also packaged into AAV particles and used to infect IB3-1 cells as a transducing virus. Under these conditions, 60 to 70% of the cells could be stably transformed to geneticin resistance. Thus, AAV transducing vectors appear to be a highly efficient delivery system for stable integration and expression of genes in cultured airway epithelial cells.

Defective regulation of outwardly rectifying Cl- channels by protein kinase A corrected by insertion of CFTR.

Cystic fibrosis (CF) is a lethal genetic disease resulting in a reduced Cl- permeability, increased mucous sulphation, increased Na+ absorption and defective acidification of lysosomal vesicles. The CF gene encodes a protein (the cystic fibrosis transmembrane conductance regulator, CFTR) that can function as a low-conductance Cl- channel with a linear current-voltage relationship whose regulation is defective in CF patients. Larger conductance, outwardly rectifying Cl- channels are also defective in CF and fail to activate when exposed either to cyclic AMP-dependent protein kinase A or to protein kinase C. The role of the outwardly rectifying Cl- channel in CF has been questioned. We report here that expression of recombinant CF genes using adeno-associated virus vectors in CF bronchial epithelial cells corrects defective Cl- secretion, that it induces the appearance of small, linear conductance Cl- channels, and restores protein kinase A activation of outwardly rectifying Cl- channels. These results re-establish an involvement of outwardly rectifying Cl- channels in CF and suggest that CFTR regulates more than one conductance pathway in airway tissues.

Microcystin-induced activation of prostaglandin synthesis and phospholipid metabolism in rat hepatocytes.

The effects of microcystin-LR, a trichothecene, T-2 and saxitoxin on membrane lipid mediators of inflammatory processes were evaluated in cultured rat hepatocytes using [(14)C]arachidonic acid. Microcystin-LR significantly stimulated the release of prostacyclin, measured as 6-keto PGF(1)alpha, by 38%, and thromboxane B(2) by 50%, in a concentration-dependent manner. The trichothecene toxin T-2 enhanced the release of prostaglandin F(2)alpha by 24% and arachidonic acid by 29%; while saxitoxin did not affect the release prostaglandins or arachidonic acid. Incorporation of arachidonic acid into the lipid pool was reduced by 47% by 1 mum microcystin-LR. Changes in the distribution of radioactivity derived from [(14)C]arachidonic acid within phospholipid classes indicated that prostaglandin formation induced by microcystin-LR was also due to the release of arachidonic acid from the phosphatidylinositol pool. No statistically significant effect of toxin was observed on the contribution of [(14)C]arachidonic acid release by other classes of phospholipids or neutral lipids. These effects may be important in the mechanism of microcystin-LR-induced toxicity in the liver.

Interaction of cyclic peptides and depsipeptides with calmodulin.

A variety of small peptides bind calmodulin (CaM) and inhibit CaM-dependent enzyme activity. The cyclic peptides cyclosporin A (CSA) and gramicidin-S (GRS) are shown to bind CaM and inhibit 3',5'-cyclic nucleotide phosphodiesterase (PDE) in a calcium-dependent manner. The cyclic peptide microcystin-LR (MLR) and the depsipeptides, valinomycin (VLM) and enniatin-B (ENB), bind to CaM and inhibit PDE activity. Spectral changes exhibited by the binding of MLR, VLM and ENB to dansyl-CaM as compared to that of CSA and GRS reflected different binding sites and/or different conformational changes. The apparent binding constants (Kd) for CaM-peptide were estimated and found to be 4.8 microM for CSA, 2.85 microM GRS, 12.99 microM MLR, 4.29 microM VLM and 41.26 microM ENB. Although these peptides did not inhibit baseline PDE activity, they did inhibit CaM-dependent PDE activity in a dose-dependent manner. Half-maximal inhibition (IC50) of PDE occurred approximately at 0.11 microM MLR; 0.45 microM GRS; and greater than 5 microM for ENB, CSA and VLM. This may be the first observation that these peptides (MLR, VLM and ENB) bind to a known cytoplasmic protein and inhibit an enzyme system dependent on that protein for optimal activity. Interaction of these peptides with CaM may be responsible for creating conformational-functional changes in CaM, thus altering the signal transduction mechanism required for CaM-dependent enzymes, such as cyclic nucleotidase, protein kinases and phospholipase A2.

Effect of antihepatotoxic agents against microcystin-LR toxicity in cultured rat hepatocytes.

Primary cultures of adult rat hepatocytes were used to investigate the effects of two putative therapeutic agents, dithioerythritol and silymarin on microcystin-LR-induced hepatotoxicity. Cell injury was assessed by the extent of cellular [14C]adenine nucleotides and lactate dehydrogenase (LDH) release into the medium and the extent of hepatocyte detachment from monolayers. Microcystin-LR (1 microM) induced a significant release of both 14C-labeled nucleotides and LDH from hepatocytes as well as significant detachment of cells from monolayers. Although both dithioerythritol (0.63-5 mM) and silymarin (25-200 microM) reduced the amount of marker release and cell detachment from microcystin-LR-treated wells, silymarin provided significantly greater protection than dithioerythritol at one-tenth the concentration. Furthermore, silymarin and dithioerythritol treatment prevented morphological deformations and detachment of cells.

Fragment A of diphtheria toxin causes pH-dependent lesions in model membranes.

Fragment A of diphtheria toxin has been shown to insert into lipid bilayers at low pH (Montecucco, C., Schiavo, G., and Tomasi, M. (1985) Biochem. J. 231, 123-128; Zhao, J.-M., and London, E. (1988) J. Biol. Chem. 263, 15369-15377). In this report, evidence is provided which demonstrates that fragment A, like diphtheria toxin, can also cause the release of a fluorescent dye (calcein) from vesicles under acidic conditions and that this release parallels fragment A insertion into the membrane. Although the permeability changes are not as large as those obtained with whole toxin (Jiang, G.-S., Solow, R., and Hu, V. W. (1989) J. Biol. Chem. 264, 13424-13429), molecular sieving experiments indicate that the lesion induced by fragment A increases in size with decreasing pH and reaches an upper limit of 30 A at pH 4.0. In addition to size differences, the lesion induced by fragment A releases calcein in a graded manner, whereas diphtheria toxin causes an all-or-none release. One possible interpretation of this result is that the fragment A lesion is transient in comparison to that induced by whole toxin. Although the molecular bases for the observed differences are not understood, these data suggest that fragment A interaction with the lipid bilayer may play a significant role in mediating its own translocation across membranes and that fragment B may aid this process by initiating, enlarging, and stabilizing the lesion formed.

Characterization of diphtheria toxin-induced lesions in liposomal membranes. An evaluation of the relationship between toxin insertion and "channel" formation.

Diphtheria toxin interaction with membranes has been studied by following the release of a fluorescent dye (calcein) encapsulated within large unilamellar vesicles. Results showed that diphtheria toxin induced temperature- as well as pH-dependent permeability changes in these model membranes. Interestingly, insertion of the "channel-forming" B domain was not sufficient for calcein release, since dye release from vesicles composed of dimyristoyllecithin:cholesterol:dicetylphosphate 4:3:0.8) was completely inhibited at low temperatures which permitted B insertion. Rather, the temperature dependence of calcein release from and A domain insertion into dimyristoyllecithin:cholesterol:dicetyl phosphate vesicles suggest some relationship between "channel formation" and fragment A translocation across membranes. However, the nature of the toxin channel is called into question by our observations that channel size, in addition to activity, was pH-dependent. On the basis of these experiments, it is proposed that the toxin "channel" is the result of localized perturbations in the lipid bilayer at the interface between lipids and inserted toxin molecules that are sufficiently large in fluid membranes at low pH to allow the translocation of fragment A across the bilayer.

Interaction of microcystin-LR with SuperChar: water decontamination and therapy.

Activated charcoal (SuperChar) has been recommended for therapeutic use against poisoning by several toxic agents, but it has not been tested against microcystin-LR toxicosis. Microcystin-LR, a cyclic heptapeptide isolated from fresh water blue-green algae, has been shown to be a potent hepatotoxin in animals and in man. Studies were performed to determine the degree of in vitro adsorption of microcystin-LR to SuperChar and to assess the efficacy of SuperChar as a therapeutic agent against microcystin-LR in vivo. Scatchard analysis of the in vitro data showed that microcystin-LR bound to SuperChar with a maximum binding capacity of 0.692 mM toxin/g SuperChar with a dissociation constant of 0.016 mM. The adsorption characteristics of microcystin-LR by SuperChar was applied successfully to the decontamination of water samples spiked with microcystin-LR. While an oral (po) dose of toxin mixed with SuperChar (0.31-0.36 g/kg) modulated the toxicity, an oral pretreatment with SuperChar did not prevent lethality induced by an oral or intraperitoneal (ip) dose of microcystin-LR in mice.

Prophylactic and therapeutic use of an anti-brevetoxin (PbTx-2) antibody in conscious rats.

A polyclonal antiserum was investigated for prophylactic and therapeutic use in the treatment of brevetoxin intoxication. Conscious, tethered male rats were pre-treated with 1 ml of anti-brevetoxin IgG (PbAb) or control IgG by a 10 min infusion, then given brevetoxin (25 micrograms/kg) by a 1 hr infusion. Rats pre-treated with control IgG demonstrated signs of brevetoxin intoxication; these signs were absent in rats pre-treated with PbAb. In therapy studies, rats were infused for 1 hr with 100 micrograms/kg brevetoxin, followed immediately by 2 ml of either PbAb or control IgG. During toxin infusion, both groups showed signs of brevetoxin intoxication. Rats treated with control antibody died within 6 hr. In rats treated with PbAb, respiratory rates began to return toward baseline almost immediately, and fewer neurological signs developed. After 24 hr, nearly all neurological signs had disappeared and both core and peripheral temperatures had returned to normal. There was a time differential between two groups of signs, suggesting high and low accessibility compartments for the antibody. These compartments probably represent central and peripheral nervous system. All animals treated with PbAb survived at least 8 days. These results suggest that PbAb has both therapeutic and prophylactic potential in the treatment of brevetoxin intoxication. Further, because of the differential in efficacy in reversing central and peripheral nervous system signs of brevetoxin intoxication, it provides useful new information on the mechanism of action of this toxin.

Fluorescence analysis of size distribution and mode of dye release from carboxyfluorescein-loaded vesicles: application to the study of complement-membrane interactions.

We wish to report a novel method for visualizing large unilamellar vesicles loaded with a fluorescent dye and for monitoring changes in the size distribution as well as state of aggregation of such dye-loaded liposomes. In addition, we demonstrate that this method can be used to distinguish between all-or-none release of dye and graded release of dye from individual vesicles. Using this technique, we have characterized complement-mediated release of carboxyfluorescein from large unilamellar vesicles and have found that C5-8 complexes mediate a graded release of dye while C5-9 complexes cause an all-or-none release. Furthermore, complement appears to preferentially attack the medium to larger-sized vesicles in our population of large unilamellar vesicles while smaller vesicles appear to be selectively spared.

Standardized sequence for carving and finishing amalgam restorations.

This method of amalgam carving and finishing, described in a step-by-step sequence, is fast and effective. The initial step of using the acorn burnisher eliminates the great bulk of excess amalgam and contours the restoration. A simple armamentarium is all that is required for carving, burnishing, and polishing. A general scheme for reestablishing original or ideal occlusal morphology has been discussed, emphasizing distinct triangular and marginal ridges and convex contour. Attention was focused on the prevention of ditching at the cavosurface margin, thus ensuring full benefit of the improved physical properties of silver amalgam.