Farnesyltransferase inhibitors induce dramatic morphological changes of KNRK cells that are blocked by microtubule interfering agents

Farnesyltransferase inhibitors (FTIs) exhibit the remarkable ability to inhibit transformed phenotypes of a variety of human cancer cell lines and to block the growth of cancer cells in a number of animal model systems. In this paper, we report that the addition of FTI to v-K-ras- transformed NRK cells (KNRK) results in dramatic morphological changes. Within 24 h after the addition of FTI, the round morphology of KNRK cells was changed to an elongated (flattened and spread out) morphology resembling those of untransformed NRK cells. No morphological effects were seen when similar concentrations of FTI were added to NRK cells. Phalloidin staining showed that FTI treatment did not restore the disrupted actin cytoskeleton in KNRK cells. In contrast, FTI addition resulted in the appearance of extensive microtubule networks in KNRK cells. The addition of a low concentration (1.2 nM) of vincristine or vinblastine, agents that interfere with microtubule dynamics, blocked the FTI-induced morphological changes in KNRK cells. In contrast, cytochalasin B, which interferes with actin polymerization, did not block the morphological changes. The FTI-induced morphological changes were associated with a decrease in the percentage of cells in S-phase, and the addition of 1.2 nM vincristine did not have additional effects on cell cycle progression. A higher concentration (12 nM) of vincristine caused synergistic effect with FTI to enrich dramatically KNRK cells in G₂/M phase. These results suggest that FTI affects cell morphology and that microtubule dynamics are involved in these processes.     

Terminal region recognition factor 1, a DNA-binding protein recognizing the inverted terminal repeats of the pGKl linear DNA plasmids.

The yeast linear DNA plasmids pGKl1 and pGKl2 contain inverted terminal repeats (ITRs) and terminal proteins covalently bound to the 5' termini of each plasmid. The presence of these features suggests a protein-primed mechanism of DNA replication, similar to that exemplified by mammalian adenovirus and phi 29 phage of Bacillus subtilis. In this paper, we report the identification of an activity in cytoplasmic extracts of yeast harboring the pGKl plasmids that recognizes the termini of both pGKl1 and pGKl2. We call this activity TRF1, for terminal region recognition factor 1. Deletion analyses and DNase I protection experiments demonstrate that the activity recognizes base pairs 107-183 within the ITR of pGKl1, and base pairs 126-179 within the ITR of pGKl2. The presence of T-tracts within these two regions, but otherwise dissimilar nucleotide sequences, suggests that TRF1 recognizes a common structural feature within the ITRs of the two plasmids. TRF1 has been partially purified from yeast cytoplasmic extracts and Southwestern analysis indicates that the apparent molecular mass of the protein is 16 kDa. By expressing three open reading frames from pGKl2 in Escherichia coli, we found that open reading frame 10 (ORF10) of pGKl2 encodes TRF1. The sequence of the ORF10 gene product indicates that TRF1 is a highly basic protein of small molecular mass. Comparison of TRF1 with other DNA-binding proteins known to recognize the terminal regions of linear DNAs, such as NFI and NFIII involved in adenovirus DNA replication, and phi 29 p6, involved in phi 29 DNA replication, indicates that TRF1 has a different mode of binding.     

Present status and issues regarding X-ray medical checkup vehicles in preventive medicine: usefulness of mass screening for lung cancer by an X-ray medical checkup vehicle

Although the prevention of habituation-related diseases has become an important topic in Japan, the early detection of cancers such as lung, gastric, and breast cancers is an important issue for X-ray-related imaging modalities. High cost-benefit and cost-effectiveness are necessary to perform mass screenings such as those for lung cancer. In order to assess cost-benefit and cost-effectiveness, a total of 100 institutions nationwide were investigated, with a 41% of recovery rate. There were at least one or two institutions in each prefecture. Cost-benefit analysis was based on factors including the price of the medical check-up vehicle, its service life, and income from the examinations. The mean price of medical check-up vehicles used for chest X-ray examinations was 4,445,000 yen. Cost-effectiveness analysis was based on the expense incurred to discover one lung cancer. According to our research, the cost-effectiveness involved in detecting one lung cancer by conventional chest X-ray examination was about 2,270,167 yen/person. Since this amount seems unduly high, it is necessary to improve cost-effectiveness.     

Loss of tuberous sclerosis complex 1 (Tsc1) expression results in increased Rheb/S6K pathway signaling important for astrocyte cell size regulation

Individuals with tuberous sclerosis complex (TSC) develop central nervous system abnormalities that may reflect astrocyte dysfunction. In an effort to model astrocyte dysfunction in TSC, we generated mice lacking Tsc1 (hamartin) expression in astrocytes and demonstrated that Tsc1-null astrocytes exhibit abnormalities in contact inhibition growth arrest. In this study, we demonstrate that hamartin-deficient astrocytes are also defective in cell size regulation. We show that the increase in Tsc1-null astrocyte size is associated with increased activation of the S6-kinase pathway. In keeping with recent reports that the hamartin/tuberin complex may regulate Rheb and downstream S6K activation, we demonstrate that expression of either Rheb or S6K in primary astrocytes results in increased S6 pathway activation, and that inhibition of Rheb activity in Tsc1-deficient astrocytes using either pharmacologic or genetic strategies markedly reduces S6 activation. Collectively, these observations suggest that TSC inactivation in astrocytes results in defective cell size regulation associated with dysregulated Rheb/mTOR/S6K pathway activity.     

MT neurons in the macaque exhibited two types of bimodal direction tuning as predicted by a model for visual motion detection

We previously proposed a model for detecting local image velocity on the magnocellular visual pathway (Kawakami & Okamoto (1996) Vision Research, 36, 117-147). The model detects visual motion in two stages using the hierarchical network that includes component and pattern cells in area MT. To validate the model, we predicted two types of bimodal direction tuning for MT neurons. The first type is characteristic of component cells. The tuning is bimodal when stimulated with high-speed spots, but unimodal for low-speed spots or for bars. The interval between the two peaks widens as the spot's speed increases. The second type is characteristic of pattern cells. The tuning is bimodal when stimulated with low-speed bars, but unimodal for high-speed bars or for spots. The interval widens as the bar's speed decreases. To confirm this prediction, we studied the change of direction tuning curves for moving spots and bars in area MT of macaque monkeys. Out of 35 neurons measured at various speeds, six component cells and four pattern cells revealed the predicted bimodal tunings. This result provided neurophysiological support for the validity of the model. We believe ours is the first systematic study that records the two types of bimodality in MT neurons.     

Transdifferentiation of the retinal pigment epithelia to the neural retina by transfer of the Pax6 transcriptional factor

The Pax6 gene plays an important role in eye morphogenesis throughout the animal kingdom. The Pax6 gene and its homologue could form ectopic eyes by targeted expression in Drosophila and Xenopus. Thus, this gene is a master gene for the eye morphogenesis at least in these animals. In the early development of the vertebrate eye, Pax6 is required for the instruction of multipotential progenitor cells of the neural retina (NR). Primitive retinal pigment epithelial (RPE) cells are able to switch their phenotype and differentiate into NR under exogenous intervention, including treatment with fibroblast growth factors (FGFs), and surgical removal of endogenous NR. However, the molecular basis of phenotypic switching is still controversial. Here, we show that Pax6 alone is sufficient to induce transdifferentiation of ectopic NR from RPE cells without addition of FGFs or surgical manipulation. Pax6-mediated transdifferentiation can be induced even at later stages of development. Both in vivo and in vitro studies show that the Pax6 lies downstream of FGF signaling, highlighting the central roles of Pax6 in NR transdifferentiation. Our results provide an evidence of retinogenic potential of nearly mature RPE and a cue for new therapeutic approaches to regenerate functional NR in patients with a visual loss.     

Initiation of DNA replication at the primary origin of bacteriophage T7 by purified proteins: requirement for T7 RNA polymerase.

The primary origin of bacteriophage T7 DNA replication is located 15% of the distance from the left end of the T7 DNA molecule. This intergenic segment is A + T-rich, contains a single gene 4 protein recognition site, and is preceded by two tandem promoters for T7 RNA polymerase [RNA nucleotidyltransferase (DNA-directed), EC 2.7.7.6]. Analysis by electron microscopy shows that T7 DNA polymerase [DNA nucleotidyltransferase (DNA-directed), EC 2.7.7.7] and gene 4 protein initiate DNA synthesis at randomly located nicks on duplex DNA to produce branched molecules. However, upon the addition of T7 RNA polymerase and ribonucleoside triphosphates 14% of the product molecules have replication bubbles, all of which are located near the primary origin observed in vivo; no such initiation occurs on T7 deletion mutant LG37 DNA, which lacks the primary origin. We have also studied initiation by using plasmids into which fragments of T7 DNA have been inserted. DNA synthesis on these templates is also dependent on the presence of T7 RNA polymerase and ribonucleoside triphosphates. DNA synthesis is specific for plasmids containing the primary origin, provided they are first converted to linear forms.