Comparison of retentive properties of four post systems. This study compared the retention of posts in the root for various diameters (1.3 mm and 1.6 mm) of three prefabricated post systems (Unity, Filpost, and Brasseler) with that of the previously reported No. 1 and No. 2 Flexi-Post systems. The experiment was divided into three groups comprising 10 samples for each post system studied. In group 2, the Filpost system was cemented both with a resin cement and zinc phosphate cement. Retention values were obtained with a modified universal testing machine. The retention of 1.3 mm posts from most to least retentive was Flexi-Post (zinc phosphate) greater than Filpost (zinc phosphate) greater than Filpost (resin) greater than Brasseler (zinc phosphate), greater than Unity (resin). The retention of 1.6 mm posts from most to least was Flexi-Post (zinc phosphate) greater than Filpost (zinc phosphate) greater than Brasseler (zinc phosphate), Unity (resin) greater than Filpost (resin). The Filpost system achieved higher retention with zinc phosphate cement than it did with resin cement.


Retentive strength, disintegration, and marginal quality of luting cements. This study evaluated the retention of complete crowns by using five different methods of cementation. Complete crowns were prepared with standardized dimensions on extracted human molars. Metal crowns were cast with a high noble gold ceramic alloy and were cemented with zinc phosphate cement, glass ionomer cement, composite resin cement, composite resin cement with a dentinal bonding agent, and adhesive resin cement. The retention was measured by subjecting the specimens to tensile load until fracture occurred. The disintegration was measured according to American Dental Association Specification No. 8, and the condition of the cements at the margins of crowns was analyzed by use of a scanning electron microscope. Kruskal-Wallis one-way analysis of variance revealed statistically significant differences between the mean retentive strengths. The retention of the zinc phosphate and the glass ionomer groups was significantly different from that of the adhesive resin group. The retention of the adhesive resin cement was 65% greater than the retention of the composite resin and the composite resin/dentinal bonding agent group, but the Mann-Whitney Wilcoxon rank sum test did not depict this difference as significant. The mean +/- SD of the disintegration for the zinc phosphate, the glass ionomer cement, and the composite resin cement was 0.025 +/- 0.013, 0.023 +/- 0.011, and 0.017 +/- 0.001, respectively. The scanning electron microscope analysis of the margins revealed that the composite resin cement was almost intact, the zinc phosphate was subjected to limited disintegration, and the glass ionomer displayed the worst marginal integrity.


The degree of zinc phosphate cement coverage of complete crown preparations and its effect on crown retention. This investigation examined the amount of retentive area covered by cement under complete coverage crowns and its effect on retention. Sixty crowns of self-cured acrylic resin were prepared on 60 identical brass dies and were divided into six groups of 10 crowns each according to different cement applications. The results showed that retention was dependent on the amount of retentive area covered by the cement. The amount of cement in the occlusal part of the cementation space did not affect retention.


Dimensional accuracy of castable apatite ceramic crowns: the influence of heat treatment on dimensional changes and distortion of crowns. Castable apatite ceramic crowns are subjected to heat treatment during crystallizing and during coloring after casting. The dimensional accuracy of the heat-treated crowns was investigated with respect to its influence on dimensional changes in castable apatite ceramic crowns and on distortion of the interior of the crowns, using two investment techniques and a three-coordinate measuring machine. Dimensional changes were not influenced by the number of coloring firings. Distortion of the interior of crowns was slightly outward at the gingival plane. Shrinkage because of crystallizing was approximately 20 microns and the shrinkage of the gingival plane was equal to that of the occlusal plane.


Thermal cycling distortion of metal ceramics: Part II--Etiology. The three-dimensional geometry of conventional fixed prostheses complicates the study of the thermal cycling distortion in metal ceramic alloys. Any explanation of the etiology of thermal cycling distortion in metal ceramic restorations must account for the observed magnitude, timing, and direction of the deformation. The simplified experimental geometry developed in Part I was applied to elucidate the etiologic factors involved in metal ceramic deformation. Techniques to minimize the thermal cycling distortion were also studied. It was found that all of the significant distortion occurred during the first thermal cycling of the alloy (oxidation) and that no distortion resulted from the application of body porcelain. The specimens that were cold worked and then oxidized had significantly more distortion than any other group. A significant reduction in distortion was observed when the initial thermal cycling was completed before the specimens were cold worked. It was determined that the release of casting- and cold working-induced stresses had a synergistic effect.


Comparison of vertical movement occurring during loading of distal-extension removable partial denture bases made by three impression techniques. Although the altered-cast impression technique attains stability and controlled tissue support for distal-extension removable partial dentures, the procedure is time-consuming and technique-sensitive. This study clinically compared the vertical displacement of distal-extension removable partial dentures made from different impression techniques. The impressions studied were the altered-cast impression, an impression made from a border-molded custom tray, and a stock tray irreversible hydrocolloid impression that served as a control. A post hoc analysis, using a Tukey Q-test, exhibited significance (p less than 0.01) between the two impression techniques after intraoral loading was performed. Although statistically significant, the 0.19 mm difference between the impression techniques may or may not be clinically relevant. Additional research is required in this area.


Evaluation of the accuracy of cast reorientation to a surveyor by prosthodontic residents. This study investigated the accuracy of a group of evaluators in reestablishing the orientation of a tripoded master cast to a surveyor instrument. Three prosthodontic residents served as evaluators to reestablish the tilt of a previously tripoded cast with three known reference coordinates. The distance from the known to the reference point was recorded for the three points in four trials by a judge with a machinist height gauge. Analysis of variance revealed a significant difference between the evaluator cast position and the known reference position. The investigation demonstrated +/- 0.2 mm errors at each of the three locations; 0.3-degree angulation errors occurred in reestablishing the tilt of the master cast in relation to the surveyor.


Masticatory ability, denture quality, and oral conditions in edentulous subjects. This study investigated the relationships between the ability of 38 patients with complete dentures to comminute a tough artificial test food and their answers to questions about the chewing experience. Also, clinical assessments were made of denture quality and oral conditions. Significant but weak relationships were found between the ability to comminute the test food and the subjective chewing experience as well as the degree of resorption of the mandibular residual ridge. Masticatory ability is conceivably determined by many other factors as well. Results indicate that dentists cannot rely on asking denture wearers about chewing problems and clinical responses with respect to oral conditions and denture quality for predicting those patients' masticatory abilities. The ability of wearers of complete dentures to comminute tough foods should be determined individually by a series of chewing tests.


The practice of complete denture prosthodontics by selected dental graduates. Graduates of a dental school in the southeastern United States were surveyed for the procedures they used in practicing complete denture prosthodontics. Of the 530 graduates representing 15 graduating classes, 80% returned the questionnaires. Most respondents (94.8%) made complete dentures, but only 3.5% devoted more than 20% of their practice time to this phase of prosthodontics. In practice, the average graduate performed approximately one half of the techniques that had been judged academically to be clinically essential. Compliance decreased proportionately with the number of years in practice. Factors affecting compliance include the influence of peer practitioners and continuing education courses. The content of the predoctoral curriculum needs to be reviewed for its relevancy to continuing education and to dental practice.


Study of the sagittal condylar path in edentulous patients. The inclination of the condylar path is a fundamental consideration in the prosthodontic treatment of the totally edentulous patient. The authors studied 55 patients at the University of the Basque Country, making three graphic registrations of the protrusive condylar movement on each side. Three examiners, working independently, used the tangential method to measure the angulation relative to Camper's plane. The results show that angulation varies greatly on each side and from one individual to another. In the treatment of edentulous patients, graphic registering techniques that can determine these variations should be used.


Standardized radiographs of the alveolar crest around implants in the mandible. The techniques currently used for standardized longitudinal radiographic evaluation of the supporting bone around dental implants are not suitable for general application. An aiming device is described for intraoral radiography used to evaluate the crestal bone height around dental implants used as retention for overdentures. This aiming device has been tested on four different implant systems by four dentists. Error analysis of serial radiographs indicates small deviations in reproducibility. It is concluded that this method is suitable for routine evaluation of dental implants.


Tissue compatibility and stability of a new zirconia ceramic in vivo. Tissue reaction and stability of partially stabilized zirconia ceramic in vivo was evaluated with the use of the subcutaneous implantation test. During the experimental period, zirconia ceramic was completely encapsulated by a thin fibrous connective tissue with less than 80 microns thickness. No changes of weight and 3-point bending strength were detected after 12 months of implantation. The result suggests that zirconia ceramic is biocompatible and no degradation of zirconia ceramic occurred.


Effects of environmental factors on maxillofacial elastomers: Part I--Literature review. Part I of a four-part report presents a comprehensive review of the literature pertaining to physical property testing of materials for facial and somatoprostheses. The report was conducted in preparation for testing four current maxillofacial elastomers: MDX 4-4210, Silastic 4-4515, Medical Adhesive type A, and Epithane-3, and two recently introduced materials: Silicone A-2186 and Silicone A-102.


Biocompatibility testing of a silicone maxillofacial prosthetic elastomer: soft tissue study in primates. Little information exists on the biocompatibility of maxillofacial prosthetic materials. Cosmesil material is a purpose-designed facial prosthetic elastomer that has an established clinical profile in humans but results of biocompatibility testing have not been published. Cosmesil, acrylic resin (positive control), black surgical gutta-percha (negative control), and Silastic 382 material (Dow Corning, Midland, Mich.) (reference control) were processed as custom-designed implants. The implants were inserted into five chacma baboons for a 12-week period in intraosseous, subperiosteal, submucosal, and intramuscular sites. The histologic assessment was based on a modified form of the FDI-ISO Technical Report 7405 for subcutaneous implants. An evaluation was made of capsule formation and inflammatory response. The statistical analysis involved a three-way ANOVA and a Tukey-Kramer Student range test. The critical level of statistical significance chosen was p less than 0.05. The study found that gutta-percha provoked a statistically significantly thicker capsule and a severe inflammatory response. Acrylic resin, Cosmesil material, and Silastic 382 material produced capsule formations and an inflammatory response that did not differ significantly. Cosmesil material is not manufactured as an implant material, but from the present findings it is considered acceptably biocompatible for its intended use where there may be contact with internal tissue spaces that are contiguous to external surfaces.


Surgical obturation using a gated prosthesis. A description is given for the fabrication of a stable, versatile surgical obturator prosthesis for the dentate patient. The wrought wire and acrylic resin prosthesis is based on the swinglock concept and allows for simple transition from wire-retained to removable obturation early in patient recovery. By using such a prosthesis, interim obturator fabrication may be delayed if the practitioner believes further healing to be desirable. In some patients, transition directly to definitive obturation may be considered.


A foam impression technique for maxillary defects. This article presents a technique used to overcome the problems of withdrawal of maxillectomy defect impressions with or without limited space as the result of trismus.


Light-cured combination obturator prosthesis. A technique for the fabrication of an open combination (hard-resilient) obturator prosthesis with VLC materials for patients undergoing partially edentulous maxillectomy is described. VLC denture resins and resilient liners are an exciting innovation in prosthodontic treatment and offer the maxillofacial prosthodontist a wider choice of treatment modalities. The technique and the resultant obturator prosthesis have the following advantages: 1. The ease and rapidity of the technique saves time for both the patient and the maxillofacial prosthodontist by introducing the open obturator prosthesis at the earliest opportunity. 2. The combination open obturator prosthesis provides a stable record base for securing jaw relation records. 3. The thickness and resiliency of the obturator prosthesis can be managed more easily. Flexibility and resiliency can be modified by the thickness of the resilient liner or by adding a rigid scaffolding according to the clinical requirements. 4. Prostheses are easy to repair or modify by using increments of VLC hard or resilient materials. 5. The combination VLC obturator prosthesis provides stability, retention, and decreased weight and obduration of residual palatal defects. 6. The technique can be used for partially edentulous or edentulous patients after maxillary resection.


Reproducibility of mandibular motion and muscle activity levels using a commercial computer recording system. A commercial computer recording system (BioPak) was tested for its accuracy and reproducibility in recording mandibular motion and muscle activity levels (EMG). Accuracy of measurements of mandibular motion was tested using sliding calipers. Accuracy of muscle activity levels was checked with a sine-wave calibration signal of known amplitude and frequency. Reproducibility of motion and of EMG was made using five control subjects each tested on 3 separate days. Computer measurements of motion were found to require correction for nonlinearity at openings beyond 45 mm. Most maximum voluntary excursions were found to be more reproducible than motions during mastication. However, lateral deviation during opening was strongly affected by magnet orientation and varied greatly between trials. Electromyographic recordings were distorted by improper treatment of high-frequency components of the signal and also varied greatly between trials. This study suggests that the BioPak system may indicate changes in some measures of mandibular motion and EMG levels in patients for whom no changes have occurred.


The use of transcranial stimulation in the fabrication of an occlusal splint. An alternative method for fabricating occlusal splints by means of electric transcortical stimulation is presented. Ensuing results in one patient suggest neuromuscular benefits. However, the centric occlusal position determined by this method is a painful procedure because of electric stimulation on the scalp. Further and extensive investigations must be carried out to assess the clinical benefits and limits of this method.


Principles for selecting interocclusal records for articulation of dentate and partially dentate casts. Minimizing the orientation error of the working and opposing casts on the articulator should reduce the extent of intraoral adjustment of restorations before their placement. This article defines and reviews principles such as the tripod of vertical support (three widely spaced occlusal contacts) and adequate horizontal stability, which enable opposing dental casts to be held together in a stable and reproducible manner at the time the mandibular cast is mounted to the articulator. During the restoration of the dentate or partially dentate patient, where restoration is to be coincident with pretreatment maximum intercuspation, the goal of the interocclusal record is to provide stability and/or support for the casts. The types of interocclusal records are organized into categories and designs based upon the remaining vertical support and horizontal stability of the dentition. The clinical indications, limitations, design, and composition of a variety of records for each of these categories are reviewed and discussed.


Comparison of bond strength of six soft denture liners to denture base resin. The bond strength of six commercial soft denture liners was evaluated using a modified tensile test. The soft denture liners investigated were Prolastic, VinaSoft, Flexor, Molloplast-B, Novus, and Super-Soft. The samples were processed according to the manufacturers' instructions to cured denture base resin (polymethyl methacrylate; PMMA). The soft denture liners were 10 x 10 x 3 mm and were processed between two PMMA blocks. The samples were placed in tension until failure. The mode of failure, cohesive or adhesive, was also recorded. The results of this study showed that the bond strength is related to the components of the materials. Prolastic, VinaSoft, and Flexor had the lowest bond strength to cured PMMA and ranged from 9.6 to 11.3 kg/cm2. Super-Soft, Novus, and Molloplast-B demonstrated better bond strengths and ranged from 16.7 to 17.6 kg/cm2. The bond strength of Novus could be improved by using the recommended bonding agent and bonded Novus at 26.1 kg/cm2 had the highest bond strength of all materials tested.


Flexible casts used in making indirect interim restorations. This article describes a procedure for making indirect interim restorations from a cast and dies made of polyvinyl siloxane impression material. The use of these flexible casts and dies facilitates the removal of the polymerized resin from the cast and the rapid set of the polyvinyl materials reduces the time involved in making the indirect interim restorations.


The usage period of dentures and their residual monomer contents. The quantity of residual monomer content in 24 maxillary dentures was detected by gas liquid chromatography. The residual monomer contents tended to be lower in dentures used for long periods than in those used for short periods. Decreases in the quantities of residual monomer in acrylic resin dentures as a result of aging can be expressed in a hyperbola. The fitted curve also suggests that most of the loss could be accomplished in 5 years; however, complete loss of the residual monomer content takes many years.


Immunosuppressive activity in the rat seminiferous tubules. Rat seminiferous tubule segments in defined stages of the epithelial cycle were isolated by transillumination-assisted microdissection. The segments were cultured together with ConA-stimulated peripheral blood lymphocytes (PBL) and incorporation of 3H-labelled thymidine was measured. Tubule segments in stages II-VIII of the seminiferous epithelial cycle inhibited PBL proliferation significantly more than stages IX-I. Inhibition was lowest in stages IX-XII and increased progressively to reach a maximum in stages II-VIII. In a more detailed analysis, tubules in stages V and VI inhibited PBL proliferation significantly less than stage II tubules. No significant difference was observed between stages II and VII. The immunosuppressive activity had molecular weights of approximately 25 kDa and approximately 65 kDa in stage II-VIII seminiferous tubules. In stage II-VI seminiferous tubules activity was present also at approximately 10 kDa. The results suggest that the seminiferous tubules produce high-molecular weight immunosuppressive activity in a stage-dependent way. In addition to its contribution to the immunologically privileged milieu of the testis this activity may also be involved in the physiological regulation of DNA synthesis in the seminiferous epithelium.


Interaction between inoculated allogeneic lymphocytes and fetal rats. Interaction between allogeneic lymphocytes and 12-20-day gestation rat fetuses was examined in vitro and in vivo. Intravenous injection of lymphocytes was achieved either by in vitro cannulation of the vitelline vein followed by culture of the whole fetus, or of its organs, or by in utero cannulation of the vitelline vein through the uterine wall. Cytotoxic reactions on the part of the inoculated allogeneic lymphocytes against fetal tissue could not be detected in the young hosts. The fetal spleen, mesentery and intestine appeared to have a specific affinity for adult lymphocytes from the earliest gestational age examined.


The influence of intravenously introduced allogeneic lymphocytes on fetal rats. Semi-allogeneic or allogeneic bone marrow cells, or allogeneic spleen cells, were injected in utero into PVG, DA and Fischer rat fetuses at 15-18 days of gestation via the vitelline vein. Prenatal (PVG x DA) F1 bone marrow cell inoculation produced allograft tolerance far more frequently in DA recipients than in PVG recipients. Inoculation of allogeneic or semi-allogeneic spleen cells into rat fetuses failed to induce allograft tolerance but sensitized the recipients. The observation that Fischer recipients could be rendered tolerant to skin allografts by prenatal inoculation of PVG, but not DA, bone marrow cells indicated that variation in susceptibility to tolerance induction among different donor-recipient strain combinations is unlikely to be explicable on the basis of strain differences in developmental maturity of the host immune system.


Preferential synthesis of asymmetric antibodies in rats immunized with paternal particulate antigens. Effect on pregnancy. The effect of immunization of female Fischer rats with particulate (spleen cells) (group I) or soluble (supernatant of disintegrated spleen cells) (group II) paternal antigens previous to mating with Buffalo rats was investigated. The percentage of asymmetric IgG molecules in the serum of rats inoculated with particulate antigens was 38% while in those injected with soluble antigens it was 29% and 28% in non-immunized animals. These percentages further increased during pregnancy to 45%, 38% and 37%, respectively. The antipaternal antibody titres, as determined by indirect immunofluorescence (IIF), was much higher in the animals immunized with particulate antigens but the effector activity, judged by complement fixation, was similar in both groups. The same values were observed at the time of mating (after 3 months of immunization) and at day 17 of pregnancy. Fetus and placenta weights and offspring survival were equally greater in group I than in group II or non-immunized rats (group III). The results obtained indicate the preferential synthesis of antipaternal IgG asymmetric antibodies in rats injected with particulate antigens previous to mating and suggests a beneficial effect of these antibodies in pregnancy.


Relative immunosuppressive activity of human seminal prostaglandins. Human seminal plasma contains uniquely high concentrations of prostaglandins of the E series which are believed to contribute to its immunosuppressive effects in vivo. In order to obtain further insight into their activity we have compared the immunosuppressive properties in vitro of PGE1, PGE2 and 19-OH PGE using three immunological systems known to be modulated by prostaglandins, namely, mitogen induced lymphocyte proliferation, IL-2 and transferrin receptor expression and NK-cell mediated cytotoxicity. These studies revealed that PGE1 and PGE2 exerted a greater immunosuppressive effect than 19-OH PGE, but considerably higher levels of 19-OH PGE in semen might contribute the majority of immunosuppressive activity in vivo. Our studies also show that the lower stability of 19-OH PGE in culture media may be responsible for its lower immunosuppressive effect observed in vitro.


Antifertility effect of active immunization with ZP4 glycoprotein family of porcine zona pellucida in hamsters. Female golden hamsters were immunized with solubilized porcine zona pellucida (s-PZP) or ZP4 glycoprotein family isolated from s-PZP by preparative SDS-PAGE. Both antigen preparations induced production of antibodies which reacted not only with porcine zona pellucida but also with the hamster zona pellucida. The hamsters immunized with solubilized porcine zona pellucida mainly produced antibodies reactive to ZP3, while the hamsters immunized with ZP4 mainly produced antibodies reactive to ZP4. The former animals became permanently infertile but the infertility in the latter animals was temporary and they became pregnant later. Histological studies revealed that the ovarian follicles in hamsters immunized with s-PZP were completely destroyed leaving only atrophic follicle-like cell clusters, while in the ovaries of hamsters immunized with ZP4 a number of small follicles with oocytes remained intact. These observations are encouraging for the further characterization of the ZP4 antigens as candidates for the development of a contraceptive vaccine.


Interleukin-3-like activity levels in pregnant women: possible modulation by progesterone. Human interleukin-3-like activity (IL-3-LA), a factor possessing similar characteristics to interleukin-3 and having clony stimulating factor (CSF) activity, has recently been defined. In the present study, IL-3-LA levels in the sera of women before and after delivery were examined. The results indicate a significant increase in IL-3-LA levels in women before delivery as compared to IL-3-LA levels after delivery or to non-pregnant healthy women. The ability of mononuclear cells from women before and after delivery to produce IL-3-LA was similar to that of mononuclear cells from cord blood. In addition, the effect of progesterone on in vitro IL-3-LA production was examined and a stimulatory dose-dependent effect was observed. These observations point to the hypothesis that during pregnancy IL-3-LA levels are modulated by progesterone. With placental loss, the IL-3-LA in the sera decreases, although the mononuclear cells previously affected by the hormone continue to produce cytokines.


Overberg research projects. XII. The efficacy of an albendazole slow-release capsule in the control of nematode parasites of sheep. Albendazole intra-ruminal slow-release capsules (SRC) administered to sheep grazing on irrigated pastures in the southern Cape Province, controlled Trichostrongylus for 91 d and Haemonchus and Teladorsagia for 61 d. Treating sheep with the SRC at 47 to 61 d intervals over a period of 10 months, significantly (P less than 0.05) reduced the infective potential of the pasture. Compared with sheep which received 2 disophenol and 3 broad spectrum anthelmintic treatments during the same period, the SRC reduced the infective potential of the pasture by 71.6%, while weekly drenching with albendazole over the same period, resulted in a significant (P less than 0.05) increase (73.4%) in the infective potential of the pasture.


Tiletamine hydrochloride in combination with zolazepam hydrochloride as an anaesthetic agent in sheep. The anaesthetic effects of Zoletil, a 1:1 combination of tiletamine and zolazepam, was evaluated in 10 sheep. The optimum dose of this preparation was found to be 12 mg kg-1. It produced rapid induction, cataleptoid anaesthesia and smooth recovery with a minimal effect on blood pressure. Individual sheep, however, displayed variable reactions to the anaesthetic. Atropine premedication at 0.04 mg kg-1 did not have any significant effect on either heart rate or blood pressure when compared to Zoletil alone.


Necropsies of eight horses infected with Strongylus equinus and Strongylus edentatus. Ponies (n = 8) approximately 18 months old, were infected with 20,000 to 30,000 infective larvae of Strongylus equinus with less than 10% contamination with Strongylus edentatus larvae and necropsied 7 months post-infection. Lesions were present in the omentum, liver, pancreas, ventral colon, caecum and occasionally in the lungs. There were numerous intraabdominal adhesions and severe multiple granulomatous omentitis. Pancreatic damage, which characterises S. equinus, was exceptionally mild and was manifested mainly by slight periductular infiltration of eosinophils. Granulomas associated with larvae were found in the connective tissue associated with the pancreas. Larvae were recovered from the flanks, the peritoneum, the caecum and the kidney. Larval recovery was low, with a high percentage of the total number of larvae recovered in some of the carcasses being S. edentatus.


Malignant mesothelioma in a seven-week-old puppy. A case of diffuse malignant peritoneal mesothelioma in a 7-week-old puppy is reported. The puppy presented with a recurrent abdominal effusion. An exploratory laparotomy revealed extensive adhesions. Necropsy findings included pleural thickening, and enlargement of the renal and anterior mediastinal lymph nodes. The microscopic features were typical of primary desmoplastic diffuse mesothelioma of the peritoneum and pleura with secondary lymph node metastasis (renal and anterior mediastinal). The tumour was classified as epithelial in type. The extent of the tumour and the distant metastasis were considered to reflect a high degree of malignancy. A congenital origin was suspected.


Cutaneous leishmaniasis in a sheep.

Cutaneous leishmaniasis in a sheep. A case of cutaneous leishmaniasis in a sheep from the Eastern Transvaal is described. The pinna of one ear was swollen and the overlying skin thickened and diffusely encrusted. Histopathological examination of a skin biopsy revealed a diffuse dermal infiltration of macrophages, plasma cells and lesser numbers of eosinophils and neutrophils, as well as a few multinucleated giant cells. Macrophages, particularly in the superficial dermis, contained large numbers of small basophilic bodies morphologically compatible with the amastigote stage of Leishmania spp. Changes in the overlying epidermis included orthokeratotic hyperkeratosis, pseudoepitheliomatous hyperplasia with a focal erosion and crust formation. No attempts were made to culture the organism.


Synthesis and biological activity of CCK heptapeptide analogues. Effects of conformational constraints and standard modifications on receptor subtype selectivity, functional activity in vitro, and appetite suppression in vivo. A series of modifications of the CCK7 analogue (des-NH2)Tyr(SO3-)-Nle-Gly-Trp-Nle-Asp-Phe-NH2 was prepared and tested for binding to guinea pig CCK-A and CCK-B receptors and in CCK-A-mediated functional assays. Selected analogues also were tested for appetite suppressant activity in rats. Several conformationally restricted residues in the C-terminal tetrapeptide region, including delta Z-Phe33, (N-Me)Phe33, (N-Me)Asp32, (N-Me)Leu31, and 3PP31 (3PP = trans-3-n-propyl-L- proline) were found to be acceptable modifications at one or both receptor subtypes. The (N-Me)Asp32 and (N-Me)Leu31 modifications afforded potent and selective CCK-A and CCK-B ligands, respectively. SAR studies in the N-terminal acyldipeptide region examined structural requirements for the side chain at position 28, where Gly and Pro replacements were found to possess high affinity at both receptor subtypes. Other conformationally restrictive modifications were less active. All of the analogues that showed high affinity (less than 10 nM) for the CCK-A receptor also were full agonists in amylase release and most were full or nearly full agonists in the phosphoinositide (PI) turnover assay, the most notable exception being the delta Z-Phe33 analogue, which showed 69% of the maximal response in the PI assay. Potent activity in suppression of food intake in rats was found for selected analogues.


Design and synthesis of some substrate analogue inhibitors of phospholipase A2 and investigations by NMR and molecular modeling into the binding interactions in the enzyme-inhibitor complex. A series of substrate analogue inhibitors of pancreatic phospholipase A2 has been designed and synthesized. The compounds were tested in a novel dual-screening system based on parallel assays with monomeric and micellar substrates. Intermolecular nuclear Overhauser effects between vinylic protons on one inhibitor and identified active site residues on the bovine pancreatic enzyme have been observed in solution NMR studies of the enzyme-inhibitor complex. It can be deduced from both the biochemical results and the NMR data that the mode of interaction between this type of inhibitor and the active site of phospholipase A2 is essentially the same, irrespective of the presence or absence of an aggregated phospholipid surface. A model of the binding between the enzyme and inhibitor which incorporates the two-dimensional NMR data has been developed. The model can account for the activity of modified inhibitor structures and can be extrapolated to an assessment of the mode of binding of the natural substrate itself.


Molecular determinants of the platelet aggregation inhibitory activity of carbamoylpiperidines. A series of alpha,alpha'-bis[3-(N,N-dialkylcarbamoyl)piperidino]-p- xylenes were synthesized and tested for their inhibitory activity on ADP-induced aggregation of human platelets. A parabolic curve was obtained when log 1/C (activity) was plotted against log P (octanol/water partition coefficient). Using this as a model, a new analogue, alpha,alpha'-bis-[3-(N-methyl-N-butylcarbamoyl)piperidino]-p-xylen e (3g), was synthesized with a predicted IC50 of 25 microM. When this compound was subsequently evaluated, the IC50 was 22.1 +/- 5.5 microM, demonstrating the applicability of this model. The amide oxygen of the carbamoyl substituent appeared necessary for activity. Thus, for example, when the amide carbonyl group of 3a (IC50 = 44.5 microM) was reduced to CH2, the resulting compound 4 had a dramatically reduced activity, IC50 = 1565 microM. Compound 3a was resolved into (+) and (-) enantiomers and a meso (0) diastereomer using fractional crystallization, diastereomeric tartrate formation, and chiral HPLC. Compared to (-)-3a, the (+) isomer was 15 times more potent when ADP was the agonist and 19 times more active when collagen was used as the agonist. Molecular modeling of R,R- and S,S-3a using the SYBYL program was used to examine their interactions with phosphatidylinositol (PI). There was a better fit between PI and the R,R-3a with the energy of interaction being 17.6 kcal/mol less than that of the S,S-3a/PI complex. Although the absolute stereochemistry of individual enantiomers is not known, this study shows that R,R-3a interacts more favorably with PI than does S,S-3a and that (+)-3a is a more potent inhibitor of human platelet aggregation than (-)-3a. It is postulated that because of their lipophilicity, these compounds penetrate the platelet membrane and are then protonated at the pH of the cytosol. The protonated N then neutralizes the anionic charge on the membrane phosphoinositides, thereby rendering them less susceptible to hydrolysis by phospholipase C. Thus, the determinant parameters for optimum antiplatelet activity in 3-carbamoylpiperidines are (1) the amide carbonyl, (2) appropriate stereochemistry of the 3-substituent and (3) a log P value of about 4.5.


DNA-directed alkylating agents. 5. Acridinecarboxamide derivatives of (1,2-diaminoethane)dichloroplatinum(II). A series of acridine-2- and -4-carboxamide-linked analogues of PtenCl2 has been prepared and evaluated for biological activity against several tumor cell lines in vitro and in vivo. The platinum complexes were generally more cytotoxic than the corresponding ligands against wild-type P388 leukemia cells in vitro, with acridine-4-carboxamide complexes being the more effective. In contrast to cisplatin and PtenCl2, the complexes were equally active in vitro against both wild-type and cisplatin-resistant P388 lines. The 4-carboxamide complexes showed high levels of in vivo activity (ILS greater than 100%) against wild-type P388 using a single-dose protocol, and one compound was also significantly active in vivo in a cisplatin-resistant line, against which cisplatin and PtenCl2 are inactive.


TSAO analogues. Stereospecific synthesis and anti-HIV-1 activity of 1-[2',5'-bis-O-(tert-butyldimethylsilyl)-beta-D-ribofuranosyl]-3'-spiro -5''- (4''-amino-1'',2''-oxathiole 2'',2''-dioxide) pyrimidine and pyrimidine-modified nucleosides. Several analogues of a new lead for anti-HIV-1 agents [1-[2',5'-bis-O-(tert-butyldimethylsilyl)-beta-D-ribofuranosyl]-thymine] -3'-spiro-5''-(4''-amino-1'',2''-oxathiole 2'',2''-dioxide) (TSAO) modified at positions N-3, O-4 and C-5 of the thymine moiety, have been prepared and evaluated as inhibitors of HIV-1 replication. A new stereoselective synthetic procedure is described. Reaction of 1,2-di-O-acetyl-5-O-benzoyl-3-C-cyano-3-O-mesyl-D-ribofuranose with pyrimidine bases, followed by treatment with Cs2CO3 afforded stereoselectively, beta-D-ribofuranosyl-3'-spiro nucleosides. 2',5'-O-Deacylation and subsequent treatment with tert-butyldimethylsilyl chloride gave the TSAO derivatives. Only those analogues having a tBDMSi group at both the C-5' and C-2' positions of the ribose moiety showed potent anti-HIV-1 activity. The activity ranged from 0.060 microM to 1.0 microM. Introduction of an alkyl or alkenyl function at N-3 of the thymine ring markedly decreased cytotoxicity without affecting the antiviral activity. While markedly active against HIV-1, the TSAO derivatives had no activity against HIV-2 or SIV. They represent the first example of nucleoside analogues with an intact ribose moiety that discriminate between HIV-1 and other retroviruses.


Template-directed design of a DNA-DNA cross-linker based upon a bis-tomaymycin-duplex adduct. A template-directed approach to the design of a DNA-DNA interstrand cross-linker based upon the structure of a bis-tomaymycin-duplex adduct has been carried out. Tomaymycin is a member of the pyrrolo[1,4]benzodiazepines antitumor antibiotics. In a previous study (F.L. Boyd et al., Biochemistry 1990, 29, 2387-2403), we have shown that two tomaymycin molecules can be covalently bound to a 12-mer duplex molecule, where the drug molecules are on opposite strands six base-pairs apart, and the stereochemistry at the drug bonding site, and orientation in the minor groove, was defined by high-field NMR. This bis-tomaymycin 12-mer duplex adduct maintains the self-complementarity of the duplex and a B-type structure. In the present study we have shown using high-field NMR that this same 12-mer sequence can be truncated by two base pairs so that the two tomaymycin-modified guanines are now only four base-pairs apart, the two species of tomaymycin molecules are still bound with the same stereochemistry and orientation, and the 10-mer duplex adduct maintains its self-complementarity. In a second 10-mer duplex we have shown that changing the bonding sequence from 5'CGA to 5'AGC does not significantly affect the structure of the bis-tomaymycin-duplex adduct. However, when the sequence is rearranged so that the drugs point in a tail-to-tail orientation rather than in the previous head-to-head configuration, there are more than one species of tomaymycin bound to DNA, and, as a consequence, the bis-tomaymycin 10-mer duplex adduct loses its self-complementarity. Last, we have used the 10-mer duplex containing the 5'CGA sequence, in which the tomaymycin molecules are oriented head to head, to design an interstrand cross-linking species in which the two drug molecules are linked together with a flexible linker molecule.


Synthesis and antifolate evaluation of 10-ethyl-5-methyl-5,10- dideazaaminopterin and an alternative synthesis of 10-ethyl-10- deazaaminopterin (edatrexate). Previous findings suggesting that 5,10-dialkyl-substituted derivatives of 5,10-dideazaaminopterin warranted study as potential antifolates prompted synthesis of 10-ethyl-5-methyl-5,10- dideazaaminopterin (12a). The key step in the synthetic route to 12a was Wittig condensation of the tributylphosphorane derived from 6-(bromomethyl)-2,4-diamino-5-methylpyrido[2,3-d]pyrimidine (7a) with methyl 4-propionylbenzoate. Reaction conditions for the Wittig condensation were developed using the tributylphosphorane prepared from 6-(bromomethyl)-2,4-pteridinediamine (7b) as a model. Each of the respective Wittig products 8a and 8b was obtained in 75-80% yield. Hydrogenation of 8a and 8b at their 9,10-double bond afforded 4-amino-4-deoxy-10-ethyl-5-methyl-5,10-dideazapteroic acid methyl ester (9a) and 4-amino-4-deoxy-10-ethyl-10-deazapteroic acid methyl ester (9b). This route to 9b intersects reported synthetic approaches leading to 10-ethyl-10-deazaaminopterin (10-EDAM, edatrexate), an agent now in advanced clinical trials. Thus the Wittig approach affords an alternative synthetic route to 10-EDAM. Remaining steps were ester hydrolysis of 9a,b to give carboxylic acids 10a,b followed by standard peptide coupling with diethyl L-glutamate to produce diethyl esters 11a,b, which on hydrolysis gave 12a and 10-EDAM (12b), respectively. The relative influx of 12a was enhanced about 3.2-fold over MTX, but as an inhibitor of dihydrofolate reductase (DHFR) from L1210 cells and in the inhibition of L1210 cell growth in vitro, this compound was approximately 20-fold less effective than MTX (DHFR inhibition, Ki = 4.82 +/- 0.60 pM for MTX, 100 pM for 12a; cell growth, IC50 = 3.4 +/- 1.0 nM for MTX, 65 +/- 18 nM for 12a).


A pentahalogenated monoterpene from the red alga Portieria hornemannii produces a novel cytotoxicity profile against a diverse panel of human tumor cell lines. A polyhalogenated acyclic monoterpene, 6(R)-bromo-3(S)-(bromomethyl)-7- methyl-2,3,7-trichloro-1-octene (1) was obtained as a major component of the organic extract of the red alga Portieria hornemannii. X-ray diffraction analysis provided the complete structure, including correct placement of the different halogen atoms and determination of the absolute stereochemistry. Detailed NMR analyses provided complete 1H and 13C assignments. Compound 1 exhibited highly differential cytotoxicity against the U.S. National Cancer Institute's new in vitro human tumor cell line screening panel; brain tumor, renal, and colon tumor cell lines were most sensitive to 1, while leukemia and melanoma lines were relatively less sensitive. A second collection of P. hornemanni yielded the novel, monocyclic 2, considerably less cytotoxic and devoid of differential activity. On the basis of its unprecedented cytotoxicity profile in the NCI primary screen, compound 1 has been selected by the NCI Decision Network Committee for preclinical drug development.


Sulfonylureas: a new class of cancer chemotherapeutic agents. This study summarizes the antitumor properties of a number of sulofenur thiophene analogs against subcutaneously implanted 6C3HED lymphosarcoma with structural modification of the aryl moiety of the sulfonamide portion of the diarylsulfonylureas. The spectrum of activity of N-(p-chlorophenyl)-N'- [(5-methoxy-2-thienyl)sulfonyl]urea in the HXGC3, VRC5, CX-1, and LX-1 cell lines is also presented.


Dispiro-1,2,4,5-tetraoxanes: a new class of antimalarial peroxides. Dispiro-1,2,4,5-tetraoxanes 2-4 were synthesized as potential peroxide antimalarial drugs. They had curative activity against Plasmodium berghei in vivo at single doses of 320 and 640 mg/kg which confirms earlier unpublished data. Moreover, artemisinin (1) and 4 had equivalent ED50's against P. berghei in vivo in the multiple-dose Thompson test; neither showed any evidence of acute toxicity at total doses of more than 12 g/kg. Dispiro-1,2,4,5-tetraoxane 4 had IC50's comparable to those of 1 against Plasmodium falciparum clones in vitro. These results confirm the potential of dispiro-1,2,4,5-tetraoxanes as a new class of inexpensive peroxide antimalarial drugs.


Thieno[2,3-b]furan-2-sulfonamides as topical carbonic anhydrase inhibitors. Novel 5-[(alkylamino)methyl]thieno[2,3-b]furan-2-sulfonamides were prepared and evaluated in vitro for inhibition of human carbonic anhydrase II (CA II) and ex vivo for their ability to inhibit Ca II in the albino rabbit eye after topical administration. Compound 11a was found to lower intraocular pressure (IOP) in both the alpha-CT ocular hypertensive albino rabbit and the normal albino rabbit, but was ineffective at lowering IOP in a hypertensive, pigmented monkey model. Since 11a was highly bound to ocular pigment, a series of less basic analogs was prepared. Examples in this series were both less extensively bound to ocular pigment and more active at reducing IOP in pigmented rabbits after topical dosing. Key examples displayed moderate reactivity toward glutathione.


PgH2 analogs as potential antiplatelet derivatives. Previous observations implicating PgH2 as a direct activator of platelets suggested that derivatives of U46619, a well-characterized TxA2 receptor agonist having structural homology with PgH2, might possess antiplatelet activity. The present work describes the synthesis of [1S-(1 alpha,2 beta,3 alpha,4 alpha)]-3-[(tetrahydropyranyloxy)methyl]- 2-[2-[(triphenylmethyl)oxy]ethyl]-5-oxabicyclo[2.2.1]heptane (14) a potentially useful intermediate for the synthesis of various epoxymethano derivatives. The latter was converted to [1S-(1 alpha,2 beta (Z),3 alpha,4 alpha)]-7-[3-[[2- [(phenylamino)carbonyl]-hydrazino]methyl]-5-oxabicylo[2.2.1]hept-2 - yl]-5-heptenoic acid (23), an epoxymethano derivative of PgH2 containing a hydrazide lower side chain as previously used in the TxA2 antagonist, SQ 29,548. The intermediate 14 was also converted to [1S-(1 alpha,2 beta (Z),3 alpha,4 alpha)]-7- [3-[(hexylamino)methyl]-5-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoic acid (25) which contained a simple aza side chain as used in earlier antagonists. Derivatives 23 and 25 appeared to be specific antagonists of the human platelet TxA2 receptor as evidenced by their inhibition of U46619 (1.5 microM) induced aggregation of human platelet rich plasma (IC50 = 22 and 7 microM, respectively), while having little effect on ADP (2 microM) induced aggregation at much higher concentrations. In addition, one of these derivatives, the bicycloamine 25, was shown to compete for [3H]U46619 binding to washed human platelets with an IC50 value of 25 microM, supporting the notion that these derivatives were acting at the thromboxane receptor. However, the potency of these derivatives was less than for previously reported TxA2 antagonists, suggesting that simple linear combinations of functionality from molecules active at the human platelet thromboxane receptor will be of limited predictive value.


Improved brain delivery of AZT using a glycosyl phosphotriester prodrug. The concentration of AZT in mice plasma and brain was measured using HPLC after an ingestion of 20 mg/kg of AZT or the molar equivalent of hexadecyl 2-(alpha-D-mannopyranosidyl)ethyl 3'-azido-3'-deoxy-5'-thymidinyl phosphate 3. The results demonstrated the promising qualities of the prodrug 3 which gave AZT-5'-phosphate as the main metabolite: the total concentration of AZT derivatives detected in brain presented a peak of 156 nmol/g (5 nmol/g for AZT) at 1 h; the half-life was about 24 h (1 h for AZT) with an AUC of 4366 nmol h/g as compared to 4 nmol h/g for AZT. The lipophilic properties of 3 were confirmed by its in vitro transport of inside synaptosomes. The derivative 2-(alpha-D-mannopyranosidyl)ethyl 3'-azido-3'-deoxy-5'-thymidinyl phosphate (2) provided also a good delivery of AZT to the central nervous system, with values intermediate between those of AZT and 3.


2,3-Epoxy-10-aza-10,11-dihydrosqualene, a high-energy intermediate analogue inhibitor of 2,3-oxidosqualene cyclase. 2,3-Epoxy-10-aza-10,11-dihydrosqualene, a high-energy intermediate analogue inhibitor of 2,3-oxidosqualene (SO) cyclase was obtained by total synthesis. This involved the preparation of three main building blocks: (1) C17 squalenoid N-methylamine, (2) 3-(diphenylphosphinoyl)propanal, and (3) 5,6-epoxy-6-methylheptan-2-one. The final stages of the reconstruction of the 6E double bond were obtained by a Wittig-Horner reaction which was modified for poorly reactive systems. This compound was designed to mimic the C-8 carbonium ion formed during SO cyclization. Its inhibitory activity on various SO cyclases was evaluated and compared with the 6 Z isomer which has an unfavorable geometry. Only isomer 6 E, the carbocation analogue, was active on SO cyclases from rat liver, pig liver, S. cerevisiae, and C. albicans microsomes, with an I50 varying from 3 to 5 microM. Both E and Z isomers were inactive on squalene epoxidase at the higher concentrations tested.


Adenosine A1 antagonists. 2. Structure-activity relationships on diuretic activities and protective effects against acute renal failure. Diuretic activities of xanthine or nonxanthine adenosine antagonists and their ameliorative effects against glycerol-induced acute renal failure in rats were investigated in order to clarify the physiological and pathological function of adenosine receptors in the kidney. Diuretic and natriuretic activities of a variety of adenosine antagonists clarified systematically for the first time that the blockade of A1 receptors is more important than that of A2 receptors in sodium and water excretion and support the hypothesis that endogenous intrarenal levels of adenosine directly enhance tubular sodium readsorption. Studies of structure-activity relationships of 8-substituted xanthines in the acute renal failure demonstrated that the activation of adenosine A1 receptor was an important factor in developing such a renal failure. A series of 8-(3-noradamantyl)xanthines exhibited the extremely potent diuretic and natriuretic activities (24; 2.5 micrograms/kg, po, the ratio of urinary excretion value in treated rats to urinary excretion value in control rats = 1.69, the ratio of Na+/K+ in treated rats to Na+/K+ in control rats = 1.76) and potent ameliorative effects against glycerol-induced acute renal failure (24; 10 micrograms/kg, ip, 55% inhibition). From our detailed studies of structure-activity relationships, we can speculate that some tissue differences of the adenosine A1 receptor might exist between kidney and brain and sites of action for adenosine antagonists could be different between two renal pharmacological assays. 1,3-Dipropyl-8-(3-noradamantyl)xanthine, KW-3902 (24), was chosen for further studies and is under development as a drug for treating the acute renal failure.


Partial purification of a potassium channel with low permeability for sodium from tonoplast membranes of Hordeum vulgare cv. Gerbel. A potassium-specific tonoplast channel was identified by reconstitution of tonoplast polypeptides into planar lipid bilayer membranes. Highly purified tonoplast membranes were solubilized in Triton X-100-containing buffer and fractionated by size-exclusion chromatography. The protein fractions were assayed for ion channel activity in a planar bilayer system, and the potassium channel was routinely recovered in specific fractions corresponding to an apparent molecular mass of 80 kDa. In symmetrical electrolyte solutions of 100 mM potassium chloride, the potassium channel had a single-channel conductance of 72 pS. Substates of the channel with conductances of 17, 33 and 52 pS were frequently observed. After identification of the channel in low or high KCl, addition of sodium acetate or sodium chloride caused only insignificant conductance changes. This result suggested that the channel was not or little permeable for sodium or chloride, whereas it had similar single-channel conductance for rubidium and caesium ions as compared with potassium ions. The channel is presumably responsible for the equilibration of potassium between the vacuole and the cytosol. The role of the channel in the physiology of the barley cell under salt stress is discussed.


Effect of high pH on the plasma membrane potential and conductance in Elodea densa. In leaves of Elodea densa the membrane potential measured in light equals the equilibrium potential of H+ on the morphological upper plasma membrane. The apoplastic pH on the upper side of the leaf is as high as 10.5-11.0, which indicates that alkaline pH induces an increased H+ permeability of the plasmalemma. To study this hypothesis in more detail we investigated the changes in membrane potential and conductance in response to alterations in the external pH from 7 (= control) to 9 or 11 under both light and dark conditions. Departing from the control pH 7 condition, in light and in dark the application of pH 9 resulted in a depolarization of the membrane potential to the Nernst potential of H+. In the light but not in the dark, this depolarization was followed by a repolarization to about -160 mV. The change to pH 9 induced, in light as well as in dark, an increase in membrane conductance. The application of pH 11, which caused a momentary hyper- or depolarization depending on the value at the time pH 11 was applied, brought the membrane potential to around -160 mV. The membrane conductance also increased, in comparison to its value at pH 7, as a result of the application of pH 11, irrespective of the light conditions.


Permeation of Ca2+ through K+ channels in the plasma membrane of Vicia faba guard cells. The whole-cell patch-clamp method has been used to measure Ca2+ influx through otherwise K(+)-selective channels in the plasma membrane surrounding protoplasts from guard cells of Vicia faba. These channels are activated by membrane hyperpolarization. The resulting K+ influx contributes to the increase in guard cell turgor which causes stomatal opening during the regulation of leaf-air gas exchange. We find that after opening the K+ channels by hyperpolarization, depolarization of the membrane results in tail current at voltages where there is no electrochemical force to drive K+ inward through the channels. Tail current remains when the reversal potential for permeant ions other than Ca2+ is more negative than or equal to the K+ equilibrium potential (-47 mV), indicating that the current is due to Ca2+ influx through the K+ channels prior to their closure. Decreasing internal [Ca2+] (Cai) from 200 to 2 nM or increasing the external [Ca2+] (Cao) from 1 to 10 mM increases the amplitude of tail current and shifts the observed reversal potential to more positive values. Such increases in the electrochemical force driving Ca2+ influx also decrease the amplitude of time-activated current, indicating that Ca2+ permeation is slower than K+ permeation, and so causes a partial block. Increasing Cao also (i) causes a positive shift in the voltage dependence of current, presumably by decreasing the membrane surface potential, and (ii) results in a U-shaped current-voltage relationship with peak inward current ca. -160 mV, indicating that the Ca2+ block is voltage dependent and suggesting that the cation binding site is within the electric field of the membrane. K+ channels in Zea mays guard cells also appear to have a Cai- and Cao-dependent ability to mediate Ca2+ influx. We suggest that the inwardly rectifying K+ channels are part of a regulatory mechanism for Cai. Changes in Cao and (associated) changes in Cai regulate a variety of intracellular processes and ion fluxes, including the K+ and anion fluxes associated with stomatal aperture change.


Inhibition of Ca-activated K+ channels from renal microvillus membrane vesicles by amiloride analogs. The effect of the K(+)-sparing diuretic amiloride and two of its hydrophobic analogs, methylisobutyl amiloride (MIA) and ethylisopropyl amiloride (EIPA), on Ca-activated K+ channels from renal microvillus membrane vesicles incorporated into planar lipid bilayers was investigated. Amiloride did not inhibit currents through Ca-activated K+ channels. MIA and EIPA, however, inhibited channel currents when added to both the internal and external solutions in concentrations between 10 and 250 microM. Furthermore, when dose-response data for channel inhibition were examined using Hill plots, Hill numbers of approximately 1.5 were found for both blockers from both sides, suggesting that the mechanism of block involves multiple inhibitory binding sites. A simple kinetic scheme is proposed that can account for the results.


Transient outwardly rectifying potassium channel in the rabbit corneal endothelium. Ionic currents from freshly dissociated rabbit corneal endothelial cells were examined using patch-clamp technology and a perforated patch technique. Whole-cell current recordings revealed a transient outward K(+)-selective current that was blockable in a dose-dependent manner by 4-aminopyridine (4-AP) and quinidine. This current is similar to the 'A'-type current present in many excitable cells and is the first reported instance of such a current in any epithelial cell type. In addition to the transient current, an outwardly rectifying nonselective cation current was also observed. This current is also blocked by quinidine. To examine the possible role of these currents in the stromal volume regulatory function of the endothelium, corneas were perfused under a specular microscope with a glutathione-bicarbonate Ringer's solution (GBR) or GBR plus either 1 mM quinidine or 10 mM 4-AP. For quinidine perfusions, control corneas swelled at a rate of 6 microns/hr, while quinidine-perfused corneas swelled at a rate of 48 microns/hr. For 4-AP perfusions, control corneas swelled at a rate of -2 microns/hr, while 4-AP perfused corneas swelled at a rate of 24 microns/hr. One possible mechanism of the stromal swelling induced by these K+ channel blockers may be the result of loss of the K+ recycling pathway necessary for proper Na+/K+ ATPase function.


Flow cytometry and sorting of amphibian bladder endocytic vesicles containing ADH-sensitive water channels. The water permeability of ADH target epithelial cells is believed to be regulated by a cycle of exo-endocytosis of vesicles containing functional water channels. These vesicles were selectively labeled in intact frog urinary bladders with an impermeant fluorescent marker, 6-carboxyfluorescein. Vesicle suspensions containing the labeled endosomes were obtained by homogenization and differential centrifugation of bladder epithelial cells. The osmotic permeability of the endocytic vesicles was measured, using a stopped-flow fluorescence technique, in the absence or in the presence of HgCl2. This permeability was found very high (500 microns/sec) and inhibited by 1 mM HgCl2 (90%), thus confirming the presence of water channels. The labeled endosomes were then separated from the other membrane vesicles by flow cytometry and sorting. Their protein content was analyzed by electrophoresis on ultrathin polyacrylamide gels. Two double bands were found at 71 and 55 kDa as well as a small band at 43 kDa. They respectively correspond to 31, 38 and 10% of the total amount of silver-stained proteins present in the sorted endosomes, while they only represent 2, 4, and less than 1% of the proteins contained in the vesicle suspension, before sorting. These highly enriched proteins (or at least one of them) are likely to be involved in the mechanism of water transport. Associated to their partial purification by differential centrifugation, the sorting of the endosomes by flow cytometry seems a good way to further characterize the water channel.


Interaction of eosinophil granule major basic protein with synthetic lipid bilayers: a mechanism for toxicity. Eosinophil granule major basic protein (MBP) is a potent toxin for mammalian cells and helminths, but the mechanism of its toxicity is not known. Here we tested whether MBP toxicity is exerted through its effect on the lipid bilayer of its targets. Liposomes prepared from synthetic phospholipids were used as targets for MBP and their properties examined by fluorescence and circular dichroism (CD) spectroscopy. MBP caused a change in the temperature transition profiles of acidic liposomes (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidyl serine or an equimolar mixture of 1,2-dimyristoyl-sn-glycero-3-phosphocholine and 1,2-dimyristoyl-sn-glycero-3-phosphatidic acid) and induced their aggregation as shown by fluorescence resonance energy transfer experiments. The CD spectra and fluorescence characteristics of MBP itself were altered by its interaction with acidic lipids. Blue shifts in the emission maxima of the Trp, and of the dimethylaminonaphthyl moiety in acrylodan-labeled MBP, and a reduction in the effectiveness of quenching of Trp fluorescence by acrylamide were observed in the presence of acidic lipids. None of these effects were noted with zwitterionic lipids. This MBP: lipid bilayer interaction resulted in fusion and lysis of liposomes as indicated by the fluorescent indicator calcein. The results demonstrate that MBP associates with acidic lipids and that it disrupts, aggregates, fuses, and lyses liposomes prepared from such lipids. Such interaction might account for its wide range of toxicity.


Single-membrane and cell-to-cell permeability properties of dissociated embryonic chick lens cells. Ion channels are believed to play an important role in the maintenance of lens transparency. In order to ascribe junctional and nonjunctional permeability properties to specific lens cell types, embryonic chick lenses were enzymatically dissociated into cell clusters, cell pairs and single cells, and both cell-to-cell and single-membrane permeability properties were characterized with the patch-clamp technique. Double patch-clamp experiments and single patch-clamp experiments with Lucifer yellow in the pipette demonstrated that the cells in the dissociated preparation were well coupled, the average conductance between pairs being 42 +/- 27 nS. Double patch-clamp experiments also revealed single cell-to-cell channel events with a predominant unitary conductance of 286 +/- 38 pS. Whole-cell measurements of surface membrane conductance indicate heterogeneity within the population of dissociated embryonic chick lens cells: 63% of the cells have a voltage-independent leak current, 14% of the cells have a potassium-selective inward-rectifier current, and 23% of the cells have a current which turns off with positive voltage on a time scale on the order of seconds. The time constant for this turnoff is voltage dependent.


Epidermal growth factor receptor: elements of intracellular communication. While EGF has an important function in cell growth regulation, the molecular mechanisms by which intracellular signal connect the EGF: receptor complex on the plasma membrane with the initiation of DNA synthesis and mitogenesis is not well understood. The discovery that rasGAP, PI-3 kinase and PLC-gamma 1 are substrates for the EGF receptor tyrosine kinase has provided a beginning in understanding the biochemistry underlying growth factor receptor transduction.


Depolarization-induced calcium release from isolated triads measured with impermeant fura-2. Depolarization-induced Ca2+ release was studied in a mixture of triads and terminal cisternae isolated from rabbit skeletal muscle. The vesicles were actively loaded with known amounts of Ca2+ in the absence of precipitating anions in a solution containing 100 mM K propionate buffer. Changes in extravesicular Ca2+ were monitored with 10 microM Fura-2 (membrane impermeant form). Ca2+ release was initiated by diluting an aliquot of the loaded vesicles into a TEACl release solution designed to maintain a constant [K+].[Cl-] product. Fast release, defined as the percentage of total Ca2+ loaded which released in less than 10 sec, occurred when extravesicular free Ca2+ was in the submicromolar range and was unaffected by 5 mM caffeine under depolarizing conditions, change in external pH to 6.5, and an increase in external Mg2+ concentration from 0.1 to 0.2 mM. Thus, the Ca2+ release measured in these studies is distinct from Ca(2+)-induced Ca2+ release. The fast release more than doubled when a greater dilution (1:20 versus 1:10) of the loaded vesicles into the release solution, which would produce a larger depolarization, was used. The percentage of loaded Ca2+ which released rapidly in a particular triad preparation was similar to the percentage of vesicles structurally coupled as visualized by electron microscopy.


Transport and membrane binding of the glutamine analogue 6-diazo-5-oxo-L-norleucine (DON) in Xenopus laevis oocytes. We have examined transport and membrane binding of 6-diazo-5-oxo-L-norleucine (DON, a photoactive diazo-analogue of glutamine) and their relationships to glutamine transport in Xenopus laevis oocytes. DON uptake was stereospecific and saturable (Vmax of 0.44 pmol/oocyte.min and a Km of 0.065 mM). DON uptake was largely Na+ dependent (80% at 50 microM DON) and inhibited (greater than 75%) by glutamine and arginine (substrates of the System B0,+ transporter) at 1 mM. Glutamine and DON show mutual competitive inhibition of Na(+)-dependent transport. Preincubation of oocytes in medium containing 0.1 mM DON for 24 or 48 hr depressed the Vmax for System B0,+ transport (as measured by Na(+)-dependent glutamine uptake), this effect was highly specific (neither D-DON nor the System B0,+ substrates glutamine and D-alanine showed any independent effect) and required Na+ ions. Glutamine (1 mM in preincubation medium) protected transport from inhibition by DON. The possibility that specific inactivation of System B0,+ by DON reflects attachment of DON to the transporter was tested by examining the binding of [14C]DON to Xenopus oocyte membranes. Oocytes incubated in 100 mM NaCl in the presence of [14C]DON for up to 48 hr showed 2.4-fold higher 14C-binding to membranes than oocytes incubated in choline chloride. Na(+)-dependent DON binding (31 +/- 11 fmol/micrograms membrane protein) was suppressed by external glutamine, arginine or alanine and was largely confined to a membrane protein fraction of 48-65 kDa (as assessed by SDS-polyacrylamide gel electrophoresis). The present studies indicate that DON and glutamine uptake in oocytes are both mediated by System B0,+ and demonstrate the DON binding to a particular membrane protein fraction is associated with inactivation of the transporter, offering the prospect of using [14C]DON as a covalent label for the transport protein in order to facilitate its isolation and subsequent biochemical characterization.


On coenzyme Q orientation in membranes: a linear dichroism study of ubiquinones in a model bilayer. A general approach is developed to interpret linear dichroism (LD) spectra of ubiquinones (Qn) in host bilayers. Information is reported in terms of guest-host mutual orientation and localization. The overall orientational anisotropy of guest ubiquinone molecules is described by a basic set of limiting orientation/localization modes. Assignments of the UV transitions of the ubiquinone chromophore were obtained by the liquid crystal-linear dichroism technique and molecular orbital (CNDO/S) calculations. The LD spectra of Qn in the bilayers provided by the lyotropic nematic mesophase exhibited by water solutions of potassium laurate and decanol were interpreted on the basis of the above assignments. The resulting experimental evidence showed a multisite distribution in the host bilayer for the aromatic heads of all the investigated Qn derivatives except Q0. The orientational distribution suggested by the LD spectra fits the solubilization model recently proposed by G. Lenaz [J. Membrane Biol. (1988) 104:193-209] for ubiquinone in lipid membranes. Within this model Qn molecules are located in the midplane and their headgroups oscillate transversally across the membrane. Q0 instead has a single site location, close to the polar bilayer interface. Experimental evidence that the headgroup carbonyls tend to grasp the polar interface of the host bilayer was also obtained. Orientation and location distributions of Qn guest molecules are therefore likely to result from the tendency of their aromatic heads to grasp the polar heads of the host bilayer and from the concurrent tendency of their chains to settle into the hydrocarbon host interior.


Uridine transport in basolateral plasma membrane vesicles from rat liver. The characteristics of uridine transport were studied in basolateral plasma membrane vesicles isolated from rat liver. Uridine was not metabolized under transport measurements conditions and was taken up into an osmotically active space with no significant binding of uridine to the membrane vesicles. Uridine uptake was sodium dependent, showing no significant stimulation by other monovalent cations. Kinetic analysis of the sodium-dependent component showed a single system with Michaelis-Menten kinetics. Parameter values were KM 8.9 microM and Vmax 0.57 pmol/mg prot/sec. Uridine transport proved to be electrogenic, since, firstly, the Hill plot of the kinetic data suggested a 1 uridine: 1 Na+ stoichiometry, secondly, valinomycin enhanced basal uridine uptake rats and, thirdly, the permeant nature of the Na+ counterions determined uridine, transport rates (SCN- greater than NO3- greater than Cl- greater than SO4(2-)). Other purines and pyrimidines cis-inhibited and trans-stimulated uridine uptake.


Molecules, fossils, and the origin of tetrapods. Since the discovery of the coelacanth, Latimeria chalumnae, more than 50 years ago, paleontologists and comparative morphologists have debated whether coelacanths or lungfishes, two groups of lobe-finned fishes, are the closest living relatives of land vertebrates (Tetrapoda). Previously, Meyer and Wilson (1990) determined partial DNA sequences from two conservative mitochondrial genes and found support for a close relationship of lungfishes to tetrapods. We present additional DNA sequences from the 12S rRNA mitochondrial gene for three species of the two lineages of lungfishes that were not represented in the first study: Protopterus annectens and Protopterus aethiopicus from Africa and Neoceratodus forsteri (kindly provided by B. Hedges and L. Maxson) from Australia. This extended data set tends to group the two lepidosirenid lungfish lineages (Lepidosiren and Protopterus) with Neoceratodus as their sister group. All lungfishes seem to be more closely related to tetrapods than the coelacanth is. This result appears to rule out the possibility that the coelacanth lineage gave rise to land vertebrates. The common ancestor of lungfishes and tetrapods might have possessed multiple morphological traits that are shared by lungfishes and tetrapods [Meyer and Wilson (1990) listed 14 such traits]. Those traits that seem to link Latimeria and tetrapods are arguably due to convergent evolution or reversals and not to common descent. In this way, the molecular tree facilitates an evolutionary interpretation of the morphological differences among the living forms. We recommended that the extinct groups of lobe-finned fishes be placed onto the molecular tree that has lungfishes and not the coelacanth more closely related to tetrapods. The placement of fossils would help to further interpret the sequence of morphological events and innovations associated with the origin of tetrapods but appears to be problematic because the quality of fossils is not always high enough, and differences among paleontologists in the interpretation of the fossils have stood in the way of a consensus opinion for the branching order among lobefinned fishes. Marshall and Schultze (1992) criticized the morphological analysis presented by Meyer and Wilson (1990) and suggest that 13 of the 14 morphological traits that support the sister group relationship of lungfishes and tetrapods are not shared derived characters. Here we present further alternative viewpoints to the ones of Marshall and Schultze (1992) from the paleontological literature. We argue that all available information (paleontological, neontological, and molecular data) and rigorous cladistic methodology should be used when relating fossils and extant taxa in a phylogenetic framework.


Telomere-associated repeats in Chironomus form discrete subfamilies generated by gene conversion. In dipteran insects the most distal telomere-associated DNA known to exist consists of long, complex tandem repeats. We have classified the 340-bp tandemly arranged repeats in Chironomus pallidivittatus. The repeats are distributed in a small number of subfamilies. One type of the repeat has the character of a master unit from which other main units can be derived usually by simple changes. The derived subfamilies contain segments that are degenerate versions of the corresponding segment in the master sequence. Such segments can also occur together in one and the same repeat unit in different combinations. There is a complete absence of subfamily-specific base variants in regions lying outside of the degenerate segments. Homogenization takes place between DNA sequences that are often smaller than a whole repeat unit. The mosaic structure of the repeat arrays suggests that gene conversion is an important force in the generation and maintenance of this family of repeats.


Essential role of duplications of short motif sequences in the genomic evolution of Bombyx mori. The Bombyx fibroin gene has a discrete mosaic structure of various repetitive sequences, which may have evolved through various repeating arrangements. Detailed sequence analysis of the fibroin gene containing coding and noncoding regions revealed that the whole sequence could be arranged as an array of short repetitive sequences. A portion of the intron of the fibroin gene is one of interspersed repetitive elements. We cloned a 1.5-kb DNA fragment of the Bombyx genome that contains interspersed elements homologous to the intron sequence. Sequence comparison between the intron and the 1.5-kb fragment shows that partial duplication has frequently occurred in evolutionary progress, and the resultant repetitive blocks of short motif sequences are abundant in the genome. These facts suggest that tandem duplication of the short motif sequence is an important rearrangement in genomic evolution of the fibroin gene.


A nematode hemoglobin gene contains an intron previously thought to be unique to plants. Hemoglobin genes from plants and animals both have a characteristic chromosomal organization. Plant hemoglobin genes contains a unique intron inserted into the heme-binding domain of exon 2. This intron has not been previously reported in animal globin genes, and its loss was hypothesized to have occurred early in the evolution of hemoglobins. We report here a unique six-intron, seven-exon internally duplicated nematode hemoglobin gene that contains an intron equivalent to the plant central intron in its first repeat. This nematode hemoglobin gene has lost both the central and the normal third intron in its second repeat. The nematode globin also contains a unique intron between its secretory peptide leader sequence and its coding sequence, which is absent in other extracellular invertebrate globin genes. Possible models to explain the head-to-tail duplication of this gene are discussed.


Evolutionary relationships of multiple alpha satellite subfamilies in the centromeres of human chromosomes 13, 14, and 21. Using Southern and in situ hybridization analyses, we have earlier defined four different subfamilies of alpha satellite DNA (designated pTRA-1, -2, -4, and -7), each of which has a unique higher order structure represented almost identically on human chromosomes 13, 14, and 21. Here we present the complete sequence of single isolates of these four subfamilies, representing approximately 12 kb of sequence information. Sequences of the individual 171-bp monomers that constitute these four subfamilies (and a fifth subfamily, Alpha-R1, that is known to be present on chromosomes 13 and 21) were compared both within and between the different clones. The results indicate that, at the level of their primary sequence, the five alpha subfamilies are characterized by structures that are as unrelated to each other as the different alpha subfamilies from other chromosomes. However, sequence comparisons between monomers of these clones indicate the possibility that pTRA-2, -4, and -1 may have arisen, at least in part, from a common ancestral alphoid sequence. We also provide evidence that exchange of pTRA-1 between nonhomologous centromeres and its homogenization throughout the population, perhaps by unequal exchange mechanisms, could have occurred after the divergence of humans and chimpanzees. The evolution of multiple alphoid subfamilies within a single centromere suggests that unequal exchange mechanisms may be restricted to specific domains. This may in turn contribute to some requirement for subregional pairing of sequences along the length of the centromeres of these chromosomes.


Interchromosomal gene conversion as a possible mechanism for explaining divergence patterns of ZFY-related genes. The divergence pattern of mammalian ZFY-related genes from human (ZFY and ZFX) and mouse (Zfy-1 and Zfx) was reexamined on the basis of nucleotide substitutions at the synonymous codon-alternating positions. It is possible to explain the unusual divergence pattern of the mammalian Y-linked ZF genes by interchromosomal gene conversion by X-linked ZF genes. Furthermore, the rates of evolution of mammalian X- and Y-linked ZF genes were shown to agree well with those expected from our model.


Relative importance of molecular, neontological, and paleontological data in understanding the biology of the vertebrate invasion of land. Meyer and Wilson's (1990) 12S rRNA phylogeny unites lungfish and tetrapods to the exclusion of the coelacanth. These workers also provide a list of morphological features shared in common between modern lungfish and tetrapods, and they conclude that these traits were probably present in their last common ancestor. However, the exquisite fossil records of the abundant extinct lungfishes and rhipidistians show that at least 13 out of Meyer and Wilson's 14 supposed ancestral traits were not present in the last common ancestor of lungfishes and tetrapods. Using extant taxa to infer ancestral morphologies is fraught with difficulties; just like molecular sequences, ancestral character states of morphological traits may be severely overprinted by subsequent modifications. Modern lungfish are air-breathing nonmarine forms, yet their Devonian forebears were marine fish that did not breathe air. Fossils dating from the time of origin of tetrapods in the Devonian offer the only hope of understanding the morphological innovations that led to tetrapods; morphological analysis of the "living fossils," the coelacanth and lungfish, only lends confusion.


Evolutionary consequences of nonrandom damage and repair of chromatin domains. Some evolutionary consequences of different rates and trends in DNA damage and repair are explained. Different types of DNA damaging agents cause nonrandom lesions along the DNA. The type of DNA sequence motifs to be preferentially attacked depends upon the chemical or physical nature of the assaulting agent and the DNA base composition. Higher-order chromatin structure, the nonrandom nucleosome positioning along the DNA, the absence of nucleosomes from the promoter regions of active genes, curved DNA, the presence of sequence-specific binding proteins, and the torsional strain on the DNA induced by an increased transcriptional activity all are expected to affect rates of damage of individual genes. Furthermore, potential Z-DNA, H-DNA, slippage, and cruciform structures in the regulatory region of some genes or in other genomic loci induced by torsional strain on the DNA are more prone to modification by genotoxic agents. A specific actively transcribed gene may be preferentially damaged over nontranscribed genes only in specific cell types that maintain this gene in active chromatin fractions because of (1) its decondensed chromatin structure, (2) torsional strain in its DNA, (3) absence of nucleosomes from its regulatory region, and (4) altered nucleosome structure in its coding sequence due to the presence of modified histones and HMG proteins. The situation in this regard of germ cell lineages is, of course, the only one to intervene in evolution. Most lesions in DNA such as those caused by UV or DNA alkylating agents tend to diminish the GC content of genomes. Thus, DNA sequences not bound by selective constraints, such as pseudogenes, will show an increase in their AT content during evolution as evidenced by experimental observations. On the other hand, transcriptionally active parts may be repaired at rates higher than inactive parts of the genome, and proliferating cells may display higher repair activities than quiescent cells. This might arise from a tight coupling of the repair process with both transcription and replication, all these processes taking place on the nuclear matrix. Repair activities differ greatly among species, and there is a good correlation between life span and repair among mammals. It is predicted that genes that are transcriptionally active in germ-cell lineages have a lower mutation rate than bulk DNA, a circumstance that is expected to be reflected in evolution. Exception to this rule might be genes containing potential Z-DNA, H-DNA, or cruciform structures in their coding or regulatory regions that appear to be refractory to repair.(ABSTRACT TRUNCATED AT 400 WORDS)


Perfluorocarbon liquid in the management of complex retinal detachments. 1. Conventional techniques of pars plana vitrectomy, retinal membrane stripping, scleral buckling, fluid-air or fluid-gas exchange, and silicone oil injection are ineffective in 25% of the complex retinal detachments reported. 2. The properties of perfluorocarbon liquid--clear, water immiscible, specific gravity twice that of water--make it an important adjunct to treatment of complex retinal detachments. 3. Of great interest is the fact that research has shown that visual outcome is significantly better for eyes using perfluorocarbon to initially flatten the retina.


Color vision testing.

Color vision testing. 1. Color deficiency occurs in about 8% of the population, due to alterations in the chemistry of one of the three receptive pigments for colored light, or the substitution of one pigment for another in the photoreceptor cones. 2. Subjects with pigment alteration can see a broad range of color; those with substitution of one pigment for another have broad areas of color perception defect. 3. The most common tests are pseudoisochromatic (color confusion) plates, designed with patterns hidden to the color deficient. Other tests use colored caps, tracing patterns, or an anomaloscope.


Radiographic manifestations of thoracic involvement by collagen vascular diseases. Collagen vascular diseases (CVDs) are a group of immunologic diseases that affect connective tissue at various locations throughout the body. The article reviews the variable radiographic findings associated with the common and less common CVDs.


Ophthalmic manifestations of the acquired immunodeficiency syndrome. 1. Ophthalmic manifestations of AIDS involve both anterior and posterior segments and can result in total loss of vision. Understanding of these ocular manifestations has become more important as this disease continues to proliferate. 2. Although the risk of infection by HIV in the ophthalmic setting is remote, ophthalmic health-care workers may come in contact with potentially infectious fluids in the course of normal ocular examination procedures. Infection control procedures are therefore recommended. 3. If health-care workers experience a possible exposure, they should consult a physician specializing in infectious diseases or internal medicine within 1 hour. Individuals should be evaluated for hepatitis B and tetanus, and considered for zidovudine chemoprophylaxis, in addition to being tested for HIV exposure.


High-resolution computed tomography scanning in the assessment of interstitial lung diseases. The development of high-resolution computed tomography (HRCT) has added a new dimension to the radiographic evaluation of interstitial lung diseases. The article reviews the techniques for HRCT, the normal anatomy seen by HRCT, and the HRCT findings of interstitial lung diseases.


Bronchoalveolar lavage in patients with connective tissue disease. Bronchoalveolar lavage (BAL) allows the recovery of cellular and fluid constituents that are derived from the epithelial surface of the lower respiratory tract. BAL fluid and cell analysis has become an important tool for understanding human pulmonary disease. Changes in the quantities and patterns of BAL cells and secretions have been described in a number of chronic lung disorders, especially the diffuse interstitial lung diseases. Specific BAL alterations have correlated with patient outcome and response to therapy. The connective tissue diseases have been associated with serious pleural and/or pulmonary pathology and may be a major cause of morbidity and mortality. BAL appears to be a useful semi-invasive tool in the evaluation and management of lung disease in patients with connective tissue diseases. The article describes the BAL findings in various connective tissue diseases and assesses the usefulness of BAL in the clinical management of patients with pulmonary complications.


Cardiovascular manifestations of connective tissue disorders. Disease processes that involve abnormalities of the connective tissue are protean in their manifestations because of the ubiquitous and crucial function that connective tissue serves in the body. Connective tissue supports of the heart and vascular structures play an integral role in normal cardiovascular performance, and it is not surprising that many connective tissue disorders produce important pathophysiologic processes that affect cardiac and vascular elements. The article focuses on the cardiac and vascular manifestations of hereditary and acquired forms of connective tissue disease. Special attention is focused on the use of imaging modalities and their role in diagnosing cardiovascular pathology produced by collagen disorders.


Thin-rim enhancement in Gd-DTPA-enhanced magnetic resonance images of tuberculoma: a new finding of potential differential diagnostic importance. The article describes rim enhancement seen on Gd-DTPA-enhanced magnetic resonance (MR) images of two patients with tuberculoma. Pathologic examination of the surgical specimens disclosed that the peripheral portion of the tuberculoma, which showed contrast enhancement, was composed mainly of a fibrous capsule and epithelioid granulomas and that the central zone, which showed no contrast enhancement, was composed of caseous necrotic material. In a retrospective study of MR images from 20 consecutive cases of surgically proven bronchogenic carcinoma, none showed a thin enhancing rim on postcontrast MR images. Although confirmation of these findings must await further experience, the postcontrast MR findings described here may prove to be of value in distinguishing tuberculoma and possibly other granulomatous lesions from bronchogenic carcinoma.


Intrapulmonary teratoma: a case report and review of the literature. Intrapulmonary teratomas are rare; only 30 cases have been reported in the world literature. These tumors are thought to originate from the third pharyngeal pouch. They occur equally in men and women and usually are diagnosed in the second to fourth decade of life. They are more often benign than malignant, although malignant lesions may have a favorable postoperative prognosis and benign lesions may exhibit high morbidity and mortality because of their size and location. These tumors present radiographically as lobulated masses that may contain calcification or peripheral collections of air. They most often occur in the upper lobes. The computed tomographic findings of intrapulmonary teratoma are less well known but have been described in two cases. An additional case is presented with a review of the literature and a tabular summary of the characteristics of this unusual entity.


Traumatic mediastinal lymphocele mimicking other thoracic injuries: case report. Thoracic duct injury and chylothorax are rare consequences of blunt thoracic trauma. A contained mediastinal lymph collection (ie, lymphocele) is rarer still. The article describes a case of posttraumatic mediastinal widening resulting from a high-speed motor vehicle accident. During the patient's radiologic assessment aortic rupture, paraspinal hematoma, esophageal injury, mediastinal tumor, and pseudomeningocele were sought and subsequently excluded. At this point a traumatic lymphocele was suggested, and the diagnosis was confirmed by computed tomography-guided percutaneous needle aspiration. The anatomy and physiology of the thoracic duct are reviewed.


Second-site suppressor mutations assist in studying the function of the 3' noncoding region of turnip yellow mosaic virus RNA. The 3' noncoding region of turnip yellow mosaic virus RNA includes an 82-nucleotide-long tRNA-like structure domain and a short upstream region that includes a potential pseudoknot overlapping the coat protein termination codon. Genomic RNAs with point mutations in the 3' noncoding region that result in poor replication in protoplasts and no systemic symptoms in planta were inoculated onto Chinese cabbage plants in an effort to obtain second-site suppressor mutations. Putative second-site suppressor mutations were identified by RNase protection and sequencing and were then introduced into genomic cDNA clones to permit their characterization. A C-57----U mutation in the tRNA-like structure was a strong suppressor of the C-55----A mutation which prevented both systemic infection and in vitro valylation of the viral RNA. Both of these phenotypes were rescued in the double mutant. An A-107----C mutation was a strong second-site suppressor of the U-96----G mutation, permitting the double mutant to establish systemic infection. The C-107 and G-96 mutations are located on opposite strands of one helix of a potential pseudoknot, and the results support a functional role for the pseudoknot structure. A mutation near the 5' end of the genome (G + 92----A), at position -3 relative to the initiation codon of the essential open reading frame 206, was found to be a general potentiator of viral replication, probably as a result of enhanced expression of open reading frame 206. The A + 92 mutation enhanced the replication of mutant TYMC-G96 in protoplasts but was not a sufficiently potent suppressor to permit systemic spread of the A + 92/G-96 double mutant in plants.


Nuclear factors that bind two regions important to transcriptional activity of the simian immunodeficiency virus long terminal repeat. Previous studies identified two regions in the U3 region of a molecular clone of simian immunodeficiency virus, SIVmac142, that are important to transcriptional activity under conditions of induction as well as basal-level expression (B. Renjifo, N. A. Speck, S. Winandy, N. Hopkins, and Y. Li, J. Virol. 64:3130-3134, 1990). One region includes the NF-kappa B binding site, while the other lies just 5' of this site between nucleotides -162 and -114 (the -162 to -114 region). The fact that the NF-kappa B site mutation attenuated transcriptional activity in uninduced T cells and fibroblasts where activated NF-kappa B would not be present suggested that a factor(s) other than NF-kappa B could be acting through this site. In this study, we have identified a factor which binds to a cis element overlapping the NF-kappa B site. This factor, which we call simian factor 3 (SF3), would play a role in regulation under conditions of basal level expression, whereas under conditions of induction, NF-kappa B would act via this region. SF3 may also bind to an element in the -162 to -114 region. In addition, we have identified two other factors that bind the -162 to -114 region. One, which we designated SF1, is a ubiquitous basal factor, and the other, SF2, is a T-cell-predominant phorbol myristate acetate-inducible factor. Through identification of nuclear factors that interact with the U3 region of the SIVmac142 long terminal repeat, we can gain insight into how this virus is transcriptionally regulated under conditions of basal-level expression as well as conditions of T-cell activation.


RNA- and DNA-binding activities in hepatitis B virus capsid protein: a model for their roles in viral replication. The hepatitis B virus capsid or core protein (p21.5) binds nucleic acid through a carboxy-terminal protamine region that contains nucleic acid-binding motifs organized into four repeats (I to IV). Using carboxy-terminally truncated proteins expressed in Escherichia coli, we detected both RNA- and DNA-binding activities within the repeats. RNA-binding and packaging activity, assessed by resolving purified E. coli capsids on agarose gels and disclosing their RNA content with ethidium bromide, required only the proximal repeat I (RRRDRGRS). Strikingly, a mutant in which four Arg residues replaced repeat I was competent to package RNA, demonstrating that Arg residues drive RNA binding. In contrast, probing immobilized core proteins with 32P-nucleic acid revealed an activity which (i) required more of the protamine region (repeats I and II), (ii) appeared to bind DNA better than RNA, and (iii) was apparently modulated by phosphorylation in p21.5 derived from Xenopus oocytes. Deletion analysis suggested that this activity may depend on an SPXX-type DNA-binding motif in repeat II. Similar motifs found in repeats III and IV may also function to bind DNA. On the basis of these observations, together with a reinterpretation of recent studies showing that capsid protein mutants cause defects in viral genome replication, we propose a model suggesting that hepadnavirus capsid proteins participate directly in the intracapsid reverse transcription of RNA into DNA. In this model, repeat I binds RNA whereas the distal repeats are progressively recruited to bind elongating DNA strands. The latter motifs may be required for replication to be energetically feasible.


Human immunodeficiency virus type 1 nef quasispecies in pathological tissue. The role of the nef gene in human immunodeficiency virus type 1 (HIV-1) infection is poorly understood. To provide a basis for studies on the role of nef in AIDS, we used targeted polymerase chain reaction amplification and DNA sequencing to determine the structure of nef genes in pathologic tissue from HIV-1-infected children and adults. We find that the nef reading frame is open in 92% of clones derived from both brain and lymphocytic tissue of children, suggesting that nef is expressed in these tissues. One HIV-1 clone, BRVA, obtained by coculture from the brain of an adult AIDS patient with progressive dementia, was previously shown to contain a duplicated region in nef. We show here that similar duplications are widespread in both adults and children with AIDS. However, coculture strongly selects against the broad spectrum of nef quasispecies found in tissue. These findings suggest functional selection for nef quasispecies in pathologic tissues during HIV-1 infection of the human host.


The nucleocapsid protein gene of bovine coronavirus is bicistronic. For animal RNA viruses that replicate through an RNA intermediate, reported examples of bicistronic mRNAs with overlapping open reading frames in which one cistron is contained entirely within another have been made only for those with negative-strand or double-stranded genomes. In this report, we demonstrate for the positive-strand bovine coronavirus that an overlapping open reading frame potentially encoding a 23-kDa protein (names the I [for internal open reading frame] protein) and lying entirely within the gene for the 49-kDa nucleocapsid phosphoprotein is expressed during virus replication from a single species of unedited mRNA. The I protein was specifically immunoprecipitated from virus-infected cells with an I-specific antipeptide serum and was shown to be membrane associated. Many features of I protein synthesis conform to the leaky ribosomal scanning model for regulation of translation. This, to our knowledge, is the first example of a bicistronic mRNA for a cytoplasmically replicating, positive-strand animal RNA virus in which one cistron entirely overlaps another.