Anti-apoptotic BCL-2 proteins govern cellular outcome following B-RAF(V600E) inhibition and can be targeted to reduce resistance.

In theory, pharmacological inhibition of oncogenic signaling is an effective strategy to halt cellular proliferation, induce apoptosis and eliminate cancer cells. In practice, drugs (for example, PLX-4032) that inhibit oncogenes like B-RAFV600E provide relatively short-term success in patients, owing to a combination of incomplete cellular responses and the development of resistance. To define the relationship between PLX-4032-induced responses and resistance, we interrogated the contributions of anti-apoptotic BCL-2 proteins in determining the fate of B-RAFV600E-inhibited melanoma cells. Although PLX-4032 eliminated B-RAFV600E signaling leading to marked cell cycle arrest, only a fraction of cells eventually underwent apoptosis. These data proposed two hypotheses regarding B-RAFV600E inhibition: (1) only a few cells generate a pro-apoptotic signal, or (2) all the cells generate a pro-apoptotic signal but the majority silences this pathway to ensure survival. Indeed, the latter hypothesis is supported by our observations as the addition of ABT-737, an inhibitor to anti-apoptotic BCL-2 proteins, revealed massive apoptosis following PLX-4032 exposure. B-RAFV600E inhibition alone sensitized cells to the mitochondrial pathway of apoptosis characterized by the rapid accumulation of BIM on the outer mitochondrial membrane, which could be functionally revealed by ABT-737 to promote apoptosis and loss of clonogenic survival. Furthermore, PLX-4032-resistant cells demonstrated collateral resistance to conventional chemotherapy, yet could be re-sensitized to PLX-4032 by BCL-2 family inhibition in vivo and conventional chemotherapies in vitro. Our data suggest that inhibiting anti-apoptotic BCL-2 proteins will enhance primary responses to PLX-4032, along with reducing the development of resistance to both targeted and conventional therapies.

Assessing the risk posed to free-living soil nematodes by a genetically modified maize expressing the insecticidal Cry3Bb1 protein.

Before pest-resistant genetically modified maize can be grown commercially, the risks for soil-beneficial, non-target organisms must be determined. Here, a tiered approach was used to assess the risk to free-living soil nematodes posed by maize genetically modified to express the insecticidal Cry3Bb1 protein (event Mon88017), which confers resistance towards western corn rootworm (Diabrotica virgifera; Coleoptera). The toxicity of purified Cry3Bb1 for the nematode Caenorhabditis elegans was determined using a bioassay and gene expression analysis. In addition, a soil toxicity test was used to assess the effects on C. elegans of rhizosphere soil obtained from plots of an experimental field grown with Mon88017, the near-isogenic cultivar, or either of two conventional cultivars. Finally, the indigenous nematode communities from the experimental field site with Mon88017 and from the control cultivars were analyzed. The results showed a dose-dependent inhibitory effect of Cry3Bb1 on the growth and reproduction of C. elegans, with EC50 values of 22.3 mg l⁻¹ and 7.9 mg l⁻¹, respectively. Moreover, Cry-protein-specific defense genes were found to be up-regulated in the presence of either Cry1Ab or Cry3Bb1. However, C. elegans was not affected by rhizosphere soils from Mon88017 compared to the control plots, due to the very low Cry3Bb1 concentrations, as indicated by quantitative analyses (< 1 ng g⁻¹ soil). Nematode abundance and diversity were essentially the same between the various maize cultivars. At the last sampling date, nematode genus composition in Bt-maize plots differed significantly from that in two of the three non-Bt cultivars, including the near-isogenic maize, but the shift in genus composition did not influence the composition of functional guilds within the nematode communities. In conclusion, the risk to free-living soil nematodes posed by Mon88017 cultivation can be regarded as low, as long as Cry3Bb1 concentrations in soil remain four orders of magnitude below the toxicity threshold.

Custom shade guide system for composite resins.

Shade guides that are included in most composite kits are not made of the actual resin material. Therefore the true shade, translucency, or opacity of the composite resin is not readily apparent with stock shade guides. Due to this fact, it has become evident that the best solution to the shade guide problem is to construct a custom shade guide from the actual resin. Until recently, fabrication of custom shade guides has been a time-consuming, inefficient task. Now, a simple and efficient method of forming accurate shade guides for composites is available. With its unique handle/template design, T-TABs enable a dentist to create a precise, labeled shade guide within minutes. There are many advantages to both dentist and patient when T-TABs are employed in the construction of custom shade guides.

Giardia lamblia: detection and characterization of calmodulin.

Calmodulin was detected in Giardia lamblia by radioimmunoassay and cyclic AMP phosphodiesterase activation. This protein was purified to apparent homogeneity by fast protein liquid chromatography with a yield of 260 ng of calmodulin/mg of protozoan protein. Purity was established by gel electrophoresis, gel filtration, and ion exchange chromatography. The parasite calmodulin has properties characteristic of calmodulin isolated from other eukaryotes, e.g., an apparent molecular weight of 16.7 kD; activation in calcium dependent manner of bovine heart cyclic nucleotide phosphodiesterase; and sensitivity to known calmodulin antagonists.

A histologic study of the width and nature of inter-radicular spaces in human adult pre-molars and molars.

Serial sections of jaw segments from a human post mortem population of 29 individuals providing 116 posterior interproximal sites were examined at the light microscopic level. Minimum inter-root distances at the locale of closest proximity ranged from more than 4 mm to less than 0.1 mm. They were located in the coronal third of pre-molars and mandibular first and second molars and the middle third of maxillary first molars and second molar interproximal spaces. Cancellous bone flanked by lamina dura was found between 89.6% of the teeth examined and was observed where inter-root distances exceeded 0.5 mm. At sites of less than 0.5 mm, cancellous bone was not observed, and, instead, adjacent laminae durae appeared fused (7.7%). Bone was not observed between root surfaces which were less than 0.3 mm apart, but in these instances the roots were connected by a periodontal ligament.

Regulatory factors specific for adult and embryonic globin genes may govern their expression in erythroleukemia cells.

In order to test if trans-acting regulatory factors specific for globin genes of the adult and embryonic stages of development exist in erythroid cells, transcriptionally active embryonic and adult globin genes on the same chromosome were transferred by cell fusion from the human leukemia cell K562 into phenotypically adult mouse erythroleukemia cells. Restriction-fragment-length polymorphisms of the K562 zeta (embryonic) globin genes were used to establish that all three copies of human chromosome 16 present in the K562 cell showed the same pattern of human globin gene expression after transfer to the mouse erythroleukemia cell. Adult (alpha) but not embryonic (zeta) human globin mRNA was detected in all nine of the independently derived mouse erythroleukemia hybrid cells, each of which contained human chromosome 16. Restriction endonuclease studies of the K562 alpha- and zeta-globin genes after transfer into the mouse erythroleukemia cell showed no evidence of rearrangements or deletions that could explain this loss of zeta-globin gene expression. These data suggest that regulation of globin gene expression in these erythroleukemia cells involves trans-acting regulatory factors specific for the adult and embryonic stages of development.

Antidepressant drugs suppress growth of the human pathogenic protozoan Giardia lamblia.

The tricyclic antidepressant drug chlorimipramine, some of its congeners, and its mammalian metabolites inhibited the growth in vitro of the human intestinal pathogen Giardia lamblia. The inhibitory action may reside in their ability to inhibit the membrane-associated ATPase characteristic of eukaryotes. Synthesis of other compounds of the tricyclic class may produce drugs of potential therapeutic use in the treatment of giardiasis and possibly other protozoal diseases.

Stannous fluoride and subgingival chlorhexidine irrigation in the control of plaque and chronic periodontitis.

The purpose of this study was to investigate the possible benefits of stannous fluoride dentifrice (SnF2) and once daily chlorhexidine (CH) in the control of chronic periodontitis, following a single course of scaling and root planing. 14 adult patients with pockets 4 mm or deeper received a thorough scaling and root planing, followed by instruction in the irrigation of those pockets with CH. They were given either SnF2-containing or a placebo dentifrice, and a new multituft toothbrush, but no instruction in routine oral hygiene. Plaque Index (PII), Sulcus Bleeding Index (SBI) and pocket depth were recorded prior to scaling on day 0. The same parameters and gingival shrinkage were recorded at weeks 1, 2, 4, 8 and 12. CH irrigation was discontinued at day 28. Both groups showed highly significant improvements in all parameters except gingival shrinkage after 28 days. PII was significantly less at all stages in the SnF2 group. It reached baseline values by day 56 in the placebo and day 84 in the SnF2 group. Periodontitis as assessed by SBI remained reduced significantly below baseline values in both groups at the end of the study. At this time there was no significant difference between the groups as regards SBI. It was concluded that a single course of thorough scaling and root planing followed by once daily subgingival irrigation with CH for 1 month, produced a reduction in periodontitis still apparent 2 months after cessation of irrigation. This regime allows a 3-month interval between oral hygiene visits. SnF2-containing dentifrice had a plaque control effect additional to that produced by the regime but had no effect on periodontitis as assessed by SBI.

Pulmonary artery occlusion due to histoplasmosis.

Histoplasma mediastinitis with complete or partial pulmonary artery obstruction due to compression and/or intraluminal granuloma was diagnosed in five patients and surgically verified in two. The patients, ages 12-27 years, had cough, dyspnea (four cases), and hemoptysis (two cases). Radionuclide imaging showed unilateral absence of pulmonary perfusion and minimal diminution of ventilation. Differentiation of this inflammatory process from other causes of ventilation-perfusion mismatch, for example, congenital hypoplasia and certain acquired causes of arterial obstruction, especially thromboembolism, may be possible by correlating radiographs, laminograms, and clinical history. Angiography can delineate the extent of perfusion impairment and may suggest its cause.

Improved routine chest radiography with a trough filter.

Use of the aluminum trough filter for phototimed posteroanterior chest radiographs at 120 kVp provides excellent detail within the parenchyma of the lung as well as greatly enhanced visualization of mediastinal structures on a single exposure. An improvement in overall diagnostic value was noted by a panel of radiologists in 87% of 50 cases compared with conventional 120 kVp technique. Dosimetric measurements of the entrance exposure indicate a 12.5% reduction over the mediastinum and a 62% reduction over the lateral lung field compared with routine nonfilter examinations. The filter is inexpensive and readily adapted to any x-ray unit and is, therefore, an attractive alternative to costly high kVp sources. Furthermore, the attenuation coefficient for calcium in bone and parenchyma remains relatively high with preservation of calcium contrast. Repeat exposures have been minimized and the need for additional projections virtually eliminated.

Use of bacteriophage transposon Mu d1 to determine the orientation for three proC-linked phosphate-starvation-inducible (psi) genes in Escherichia coli K-12.

We have previously used the bacteriophage transposon Mu d1 (which encodes the lacZY structural genes but without their promoter) to construct strains that have lacZY fused to phosphate-regulated promoters in Escherichia coli K-12. Among 18 identified phosphate-starvation-inducible (psi) genes, three (the phoA and two new genes: psiF and psiG) are closely linked to the proC region. The gene order (clockwise) is phoA psiF proC psiG phoB phoR. Using these mutants containing Mu d1 insertions, we devised and tested a new method to determine their orientation. In this procedure, mutants with deletions that are selectable by their ability to grow at 42 degrees C are tested for the presence of Mu d1 and of neighboring genes. Some difficulties arose during analysis of suspected deletion-containing strains derived from Mu d1 lysogens (which also contained a Tn5 element) that were caused by Mu d1 and transposon transpositions and other possible genome rearrangements. Nevertheless, we have shown that the phoA and psiF genes are transcribed clockwise and the psiG gene is transcribed counterclockwise towards proC. Because phoA, but not psiF, gene expression requires the phoB+ (positive regulator) gene product, the phoA and psiF genes do not constitute an operon. On the other hand, the psiG:lacZ fusion-bearing strain may have a fusion to the promoter-distal end of the phoB gene. This implies that phoB expression is phosphate regulated. We believe that this method may be useful in general to elucidate the direction of gene transcription.

Asymmetric prostatic hyperplasia simulating bladder tumor.

A rare case of hyperplasia of a single lateral lobe of the prostate presenting as a solitary intravesical filling defect is presented.

Fibrous mediastinitis: a late manifestation of mediastinal histoplasmosis.

Fibrosing mediastinitis is an uncommon manifestation of histoplasmosis that occurs primarily in younger patients. Findings include tracheobronchial narrowing, compromise of the main pulmonary artery and veins, and circumferential narrowing of the esophagus. The initial chest radiograph is often diagnostic, although additional studies including tomography and angiography help clarify the extent and severity of the disease process.

Displacement of pontine arteries by fungating pontine astrocytomas.

Pontine astrocytomas may fungate, encircling the basilar artery and pontomesencephalic vein and intruding between these structures and the clivus. The tumor thus may prevent anterior displacement of these vascular structures against the clivus or produce paradoxical posterior displacement. However, the pontine arteries are usually swept forward by the tumor nodules to lie anterior to the basilar artery. These arteries are also stretched as they encircle the enlarged pons, losing their normal undulations. An increased posterior extent of the vessels may be seen, presumed due to hypertrophy or displacement or both. These findings seem highly specific for the diagnosis of fungating pontine astrocytoma.