Rapamycin-sensitive mechanisms confine the growth of fission yeast below the temperatures detrimental to cell physiology.

Cells cease to proliferate above their growth-permissible temperatures, a ubiquitous phenomenon generally attributed to heat damage to cellular macromolecules. We here report that, in the presence of rapamycin, a potent inhibitor of Target of Rapamycin Complex 1 (TORC1), the fission yeast Schizosaccharomyces pombe can proliferate at high temperatures that usually arrest its growth. Consistently, mutations to the TORC1 subunit RAPTOR/Mip1 and the TORC1 substrate Sck1 significantly improve cellular heat resistance, suggesting that TORC1 restricts fission yeast growth at high temperatures. Aiming for a more comprehensive understanding of the negative regulation of high-temperature growth, we conducted genome-wide screens, which identified additional factors that suppress cell proliferation at high temperatures. Among them is Mks1, which is phosphorylated in a TORC1-dependent manner, forms a complex with the 14-3-3 protein Rad24, and suppresses the high-temperature growth independently of Sck1. Our study has uncovered unexpected mechanisms of growth restraint even below the temperatures deleterious to cell physiology.

TOR inactivation triggers heterochromatin formation in rDNA during glucose starvation.

In response to environmental cues, such as nutrient starvation, living organisms modulate gene expression through mechanisms involving histone modifications. Specifically, nutrient depletion inactivates the TOR (target of rapamycin) pathway, leading to reduced expression of ribosomal genes. While these regulatory mechanisms are well elucidated in budding yeast Saccharomyces cerevisiae, their conservation across diverse organisms remains unclear. In this study, we demonstrate that fission yeast Schizosaccharomyces pombe cells repress ribosomal gene transcription through a different mechanism. TORC1, which accumulates in the rDNA region, dissociates upon starvation, resulting in enhanced methylation of H3K9 and heterochromatin formation, facilitated by dissociation of the stress-responsive transcription factor Atf1 and accumulation of the histone chaperone FACT. We propose that this mechanism might be adapted in mammals that possess Suv39H1 and HP1, which are absent in budding yeast.

PAR2-dependent phosphorylation of TRPV4 at the trigeminal nerve terminals contributes to tongue cancer pain.

OBJECTIVE: This study aimed to clarify the interactions between the tongue and primary afferent fibers in tongue cancer pain. METHODS: A pharmacological analysis was conducted to evaluate mechanical hypersensitivity of the tongues of rats with squamous cell carcinoma (SCC). Changes in trigeminal ganglion (TG) neurons projecting to the tongue were analyzed using immunohistochemistry and western blotting. RESULTS: SCC inoculation of the tongue caused persistent mechanical sensitization and tumor formation. Trypsin expression was significantly upregulated in cancer lesions. Continuous trypsin inhibition or protease-activated receptor 2 (PAR2) antagonism in the tongue significantly inhibited SCC-induced mechanical sensitization. No changes were observed in PAR2 and transient receptor potential vanilloid 4 (TRPV4) levels in the TG or the number of PAR2-and TRPV4-expressing TG neurons after SCC inoculation. In contrast, the relative amount of phosphorylated TRPV4 in the TG was significantly increased after SCC inoculation and abrogated by PAR2 antagonism in the tongue. TRPV4 antagonism in the tongue significantly ameliorated the mechanical sensitization caused by SCC inoculation. CONCLUSIONS: Our findings indicate that tumor-derived trypsin sensitizes primary afferent fibers by PAR2 stimulation and subsequent TRPV4 phosphorylation, resulting in severe tongue pain.

Effects of three microtubule-associated proteins (MAP2, MAP4, and Tau) on microtubules' physical properties and neurite morphology.

The physical properties of cytoskeletal microtubules have a multifaceted effect on the expression of their cellular functions. A superfamily of microtubule-associated proteins, MAP2, MAP4, and tau, promote the polymerization of microtubules, stabilize the formed microtubules, and affect the physical properties of microtubules. Here, we show differences in the effects of these three MAPs on the physical properties of microtubules. When microtubule-binding domain fragments of MAP2, tau, and three MAP4 isoforms were added to microtubules in vitro and observed by fluorescence microscopy, tau-bound microtubules showed a straighter morphology than the microtubules bound by MAP2 and the three MAP4 isoforms. Flexural rigidity was evaluated by the shape of the teardrop pattern formed when microtubules were placed in a hydrodynamic flow, revealing that tau-bound microtubules were the least flexible. When full-length MAPs fused with EGFP were expressed in human neuroblastoma (SH-SY5Y) cells, the microtubules in apical regions of protrusions expressing tau were straighter than in cells expressing MAP2 and MAP4. On the other hand, the protrusions of tau-expressing cells had the fewest branches. These results suggest that the properties of microtubules, which are regulated by MAPs, contribute to the morphogenesis of neurites.

Autotoxin-mediated latecomer killing in yeast communities.

Cellular adaptation to stressful environments such as starvation is essential to the survival of microbial communities, but the uniform response of the cell community may lead to entire cell death or severe damage to their fitness. Here, we demonstrate an elaborate response of the yeast community against glucose depletion, in which the first adapted cells kill the latecomer cells. During glucose depletion, yeast cells release autotoxins, such as leucic acid and L-2keto-3methylvalerate, which can even kill the clonal cells of the ones producing them. Although these autotoxins were likely to induce mass suicide, some cells differentiated to adapt to the autotoxins without genetic changes. If nondifferentiated latecomers tried to invade the habitat, autotoxins damaged or killed the latecomers, but the differentiated cells could selectively survive. Phylogenetically distant fission and budding yeast shared this behavior using the same autotoxins, suggesting that latecomer killing may be the universal system of intercellular communication, which may be relevant to the evolutional transition from unicellular to multicellular organisms.

Facultative heterochromatin formation in rDNA is essential for cell survival during nutritional starvation.

During the cellular adaptation to nutrient starvation, cells temporarily decelerate translation processes including ribosomal biogenesis. However, the mechanisms repressing robust gene expression from the ribosomal gene cluster (rDNA) are unclear. Here, we demonstrate that fission yeast cells facing glucose starvation assemble facultative heterochromatin in rDNA leading to its transcriptional repression. Glucose starvation induces quick dissociation of the ATF/CREB-family protein Atf1 from rDNA, where in turn the histone chaperone FACT is recruited to promote H3K9 methylation and heterochromatinization. We also identify the histone acetyltransferase Gcn5 as a repressor of rDNA heterochromatinization in glucose-rich conditions, and this protein dissociates from rDNA upon glucose starvation. Facultative heterochromatin formation in rDNA requires histone deacetylases Clr3 and both the RNAi-dependent and -independent gene silencing pathways. This is essential in adaptation to starvation since mutants lacking heterochromatin formation in rDNA lead to untimely cell death during glucose starvation.

TAQing2.0 for genome reorganization of asexual industrial yeasts by direct protein transfection.

Genomic rearrangements often generate phenotypic diversification. We previously reported the TAQing system where genomic rearrangements are induced via conditional activation of a restriction endonuclease in yeast and plant cells to produce mutants with marked phenotypic changes. Here we developed the TAQing2.0 system based on the direct delivery of endonucleases into the cell nucleus by cell-penetrating peptides. Using the optimized procedure, we introduce a heat-reactivatable endonuclease TaqI into an asexual industrial yeast (torula yeast), followed by a transient heat activation of TaqI. TAQing2.0 leads to generation of mutants with altered flocculation and morphological phenotypes, which exhibit changes in chromosomal size. Genome resequencing suggested that torula yeast is triploid with six chromosomes and the mutants have multiple rearrangements including translocations having the TaqI recognition sequence at the break points. Thus, TAQing2.0 is expected as a useful method to obtain various mutants with altered phenotypes without introducing foreign DNA into asexual industrial microorganisms.

Effect of low-intensity pulsed ultrasound on orofacial sensory disturbance following inferior alveolar nerve injury: Role of neurotrophin-3 signaling.

Percutaneous treatment of low-intensity pulsed ultrasound (LIPUS) to the site of inferior alveolar nerve (IAN) transection promotes functional regeneration, but the detailed mechanism is unknown. We examined the involvement of neurotrophin-3 (NT-3), which primarily binds with tropomyosin receptor kinase C (TrkC), in functional transected IAN regeneration following LIPUS treatment in rats. Daily LIPUS treatment to the transected IAN was performed, and the mechanical sensitivity of the facial skin was measured for 14 d. On day 5 after IAN transection, the expression of NT-3 in the transected IAN and TrkC-positive trigeminal ganglion neurons were immunohistochemically examined. Further, the effect of TrkC neutralization on the acceleration of facial mechanosensory disturbance restoration due to LIPUS treatment was analyzed. LIPUS treatment to the site of IAN transection significantly facilitated functional recovery from sensory disturbance on facial skin. Schwann cells in the transected IAN expressed NT-3, and LIPUS treatment increased the amount of NT-3. The facilitated recovery from the mechanosensory disturbance by continuous LIPUS treatment was inhibited by the ongoing TrkC neutralization at the IAN transection site. These results suggest that LIPUS treatment accelerates the recovery of orofacial mechanosensory function following IAN transection through the enhancement of NT-3 signaling in the transected IAN.

NEK6-mediated phosphorylation of human TPP1 regulates telomere length through telomerase recruitment.

Shelterin component TPP1 plays critical roles in chromosome end protection and telomere length regulation. Specifically, TPP1 contains an OB-fold domain that provides an interface to recruit telomerase. However, it remains largely unknown how telomerase recruitment is regulated by cell cycle regulators. We show that TPP1 interacts with the cell cycle regulator kinase NEK6 in human cells. We found that NEK6-mediated phosphorylation of TPP1 Ser255 in G2/M phase regulates the association between telomerase activity and TPP1. Furthermore, we found evidence that POT1 negatively regulates TPP1 phosphorylation because the level of Ser255 phosphorylation was elevated when telomeres were elongated by a POT1 mutant lacking its OB-fold domains. Ser255 is located in the intervening region between the telomerase-recruiting OB-fold and the POT1 recruitment domains. Ser255 and the surrounding amino acids are conserved among vertebrates. These observations suggest that a region adjacent to the OB-fold domain of TPP1 is involved in telomere length regulation via telomerase recruitment.

Breast cancer associated with mammary hamartoma.

Mammary hamartoma is an infrequent, nonmalignant lesion. Only 12 cases of carcinomas associated with a hamartoma have been previously documented in the literature. We describe a case of invasive ductal carcinoma arising in the mammary hamartoma during the follow-up period of a previously diagnosed hamartoma.

Ionic liquid-in-oil microemulsion as a potential carrier of sparingly soluble drug: characterization and cytotoxicity evaluation.

Pharmaceutical industries have posed challenges in the topical and transdermal administration of drugs which are poorly soluble or insoluble in water and most of organic solvents. In an approach to overcome this limitation, ionic liquid-in-oil (IL/o) microemulsions (MEs) were employed to increase the solubility of a sparingly soluble drug to enhance its topical and transdermal delivery. The formulation of MEs was composed of a blend of nonionic surfactants, polyoxyethylene sorbitan monooleate (Tween-80) and sorbitan laurate (Span-20), isopropyl myristate (IPM) as an oil phase, and IL [C(1)mim] [(CH(3)O)(2)PO(2)] (dimethylimidazolium dimethylphosphate) as a pseudophase. Among various weight ratios of Tween-80 to Span-20 investigated in the ME systems, the ratio 3:2 showed excellent solubility and skin permeation enhancing effect for acyclovir (ACV) used as a model sparingly soluble drug. The size and size distribution of the ME droplets with and without drug were determined by dynamic light scattering. The permeability study of ACV incorporated in IL droplets as well as other formulations was performed into and across the Yucatan micropig (YMP) porcine skin, and the use of IL/o MEs has been shown to dramatically increase ACV administration. Finally, the cytotoxicity of the new carrier was evaluated in vitro using the reconstructed human epidermal model LabCyte™ EPI-MODEL12. It was found that the cell viability of IL/o MEs containing 4wt% IL was over 80% compared to Dulbecco's Phosphate-Buffered Salines, indicating low cytotoxicity of the carrier. Taken together these results, it can be assumed that IL-assisted nonaqueous ME could serve as a versatile and efficient nanodelivery system for insoluble or sparingly soluble drug molecules that require solubilizing agents for delivery.

Ionic liquid-assisted transdermal delivery of sparingly soluble drugs.

We report the first successful application of a novel IL-assisted non-aqueous microemulsion stabilized by a blend of two nontoxic surfactants, polyoxyethylene sorbitan monooleate (Tween-80), and sorbitan laurate (Span-20) for transdermal delivery of acyclovir, which is insoluble or sparingly soluble in water and most common organic liquids.

RPA-like mammalian Ctc1-Stn1-Ten1 complex binds to single-stranded DNA and protects telomeres independently of the Pot1 pathway.

Budding yeast Cdc13, Stn1, and Ten1 form the CST complex to protect telomeres from lethal DNA degradation. It remains unknown whether similar complexes are conserved in higher eukaryotes or not. Here we isolated mammalian STN1 and TEN1 homologs and CTC1 (conserved telomere maintenance component 1). The three proteins contain putative OB-fold domains and form a complex called CST, which binds to single-stranded DNA with high affinity in a sequence-independent manner. CST associates with a fraction of telomeres consistently during the cell cycle, in quiescent cells and Pot1-knockdown cells. It does not colocalize with replication foci in S phase. Significant increases in the abundance of single-stranded G-strand telomeric DNA were observed in Stn1-knockdown cells. We propose that CST is a replication protein A (RPA)-like complex that is not directly involved in conventional DNA replication at forks but plays a role in DNA metabolism frequently required by telomeres.

Bone regeneration induced by adenoviral vectors carrying til-1/Cbfa1 genes implanted with biodegradable porous materials in animal models of osteonecrosis of the femoral head.

A new approach for bone regeneration is needed for idiopathic osteonecrosis of the femoral head (ION). Core binding factor alpha1 (Cbfa1) was reported in 1997 as the most important transcription factor for osteoblastic differentiation. The transgenics of transcription factors affecting bone formation might be useful tools for the bone regeneration. The purpose of this study was to investigate the effects of the implantation of adenoviral vectors carrying Cbfa1 genes implanted with biodegradable porous materials on bone formation in an animal model of ION. Robust and rapid bone regeneration in large bone defects was achieved with the implantation of adenoviral vectors carrying Cbfa1 genes. These results suggest that the Cbfa1 genes induce a rapid osteoblastic differentiation and the biodegradable scaffold successfully functioned as a delivery vehicle for the Cbfa1 gene, as they induced osteogenic repair in vivo, even in necrotic bone.

Mandibular advancement improves the laryngeal view during direct laryngoscopy performed by inexperienced physicians.

BACKGROUND: When oral or nasal fiberoptic laryngoscopy is attempted, mandibular advancement has been reported to improve the laryngeal view. The authors hypothesized that mandibular advancement may also improve the laryngeal view during direct laryngoscopy. METHODS: Forty patients undergoing elective surgery under general anesthesia were included in this study. After establishment of an adequate level of anesthesia and muscle relaxation, direct laryngoscopy was performed by inexperienced physicians. Four different maneuvers--simple direct laryngoscopy without any assistance (C), simple direct laryngoscopy with mandibular advancement (M), simple direct laryngoscopy with the BURP maneuver (backward, upward, rightward pressure maneuver of the larynx; B), and simple direct laryngoscopy with both mandibular advancement and the BURP maneuver (BM)--were attempted in each subject, and the laryngeal aperture was videotaped with each procedure. An instructor in anesthesiology who was blinded to the procedure evaluated the visualization by reviewing videotape off-line, using the Cormack-Lehane classification system (grades I-IV) and a rating score within each subject (1 = best view; 4 = poorest view). The Friedman test followed by the Student-Newman-Keuls test was performed for statistical comparison. P < 0.05 was considered significant. RESULTS: The laryngeal view was improved with M and B when compared with C (P < 0.05 by both rating and Cormack-Lehane evaluation). BM was the most effective method to visualize the laryngeal aperture (P < 0.05, vs. B and M by rating evaluation), whereas B and M were the second and the third most effective methods, respectively. No statistical difference was observed between B and M with the Cormack-Lehane classification. CONCLUSION: Mandibular advancement improves the laryngeal view during direct laryngoscopy performed by inexperienced physicians.

Lysophospholipase A activity of Pseudomonas aeruginosa type III secretory toxin ExoU.

Acute lung injury in Pseudomonas aeruginosa pneumonia depends primarily on ExoU that is delivered directly into the eukaryotic cell via the type III secretion system. Recent studies demonstrated that ExoU has lipase activity, and that the cytotoxicity of ExoU is dependent on its patatin-like phospholipase domain. We investigated the phospholipase A (PLA) activity of ExoU. ExoU, but not non-catalytic ExoU-S142A, preincubated with the BEAS-2B cell lysate showed a weak increase of Ca(2+)-independent PLA(2) activity. When activated ExoU was mixed with secretory type PLA(2), more phospholipase activity was observed, suggesting that ExoU has lysophospholipase A (lysoPLA) activity. A significant increase in lysoPLA activity was also observed. Glycerol enhanced this activity and inhibitors of iPLA(2) suppressed ExoU's lysoPLA activity. Our results suggest that ExoU has a potent lysoPLA activity that requires the presence of the catalytically active site Ser(142) with an unknown eukaryotic cell factor(s) for its activation.

Clinical study on the metastasis to the eyes from breast cancer.

In our hospital, 24 patients who underwent surgery for breast cancer during 1980 to 2001 were diagnosed with metastasis to the eye. Metastasis to the choroid was found most frequently, making the choroid the most common site of metastasis. A few patients had metastasis to the orbit. Decreased visual acuity and tunnel vision were frequently found in patients who had metastasis to the choroid, and ocular floaters and blurred vision were also found in a few patients. Patients with metastasis to the orbit showed diplopia caused by ocular dyskinesia and eyelid swelling. The mean postoperative period until the diagnosis with metastasis to the eye was 3 years and 2 months, with most cases diagnosed between 20 and 40 months postoperatively, a relatively long period. We performed radiotherapy in 21 of the 24 patients, and more than half of the patients showed improvement. The mean survival period after diagnosis with metastasis to the eye was 10 months, and some of them already had recurrence to other organs such as the bones or lungs. Examination with consideration of metastasis to the eyes is required to improve the quality of life of cancer patients,.

Pseudomonas aeruginosa causes acute lung injury via the catalytic activity of the patatin-like phospholipase domain of ExoU.

OBJECTIVE: Acute lung injury in Pseudomonas aeruginosa pneumonia depends primarily on ExoU toxin being delivered directly into the eukaryotic cell cytosol through the type III secretion system. The amino-acid sequence of ExoU has a potato patatin-like phospholipase domain, similar to the sequence of mammalian Ca-independent phospholipase A2. We examined whether the acute lung injury caused by cytotoxic P. aeruginosa was dependent on the patatin-like phospholipase domain of ExoU. DESIGN: Laboratory investigation using an established mouse model for P. aeruginosa pneumonia with quantitative measurements of acute lung injury and mortality. SETTING: University experimental research laboratory. SUBJECTS: Balb/c mice. INTERVENTIONS: First, a site-directional mutation was introduced in the predicted catalytically active site of the patatin-like phospholipase domain of recombinant ExoU protein. The effect of the mutation on the catalytic activity of ExoU was tested by the in vitro lysophospholipase A assay. Second, the same site-directional mutation was introduced into the exoU gene of P. aeruginosa PA103. Mice were intratracheally infected with either a wild-type P. aeruginosa strain PA103 or an isogenic mutant containing the mutation in exoU. Acute epithelial lung injury, lung edema, bacteremia, and mortality were evaluated quantitatively. MEASUREMENTS AND MAIN RESULTS: Recombinant ExoU had lysophospholipase A activity. Site-directional mutations in the predicted catalytic site of ExoU caused a loss of the lysophospholipase A activity. Whereas the airspace instillation of PA103 caused acute lung injury and death of the infected mice, the airspace instillation of isogenic mutants secreting catalytically inactive ExoU were noncytotoxic and did not cause acute lung injury or death of the infected mice. CONCLUSION: Virulent P. aeruginosa causes acute lung injury and death by the cytotoxic activity derived from the patatin-like phospholipase domain of ExoU.