Compounds in cigarette smoke induce EGR1 expression via the AHR, resulting in apoptosis and COPD.

Chronic obstructive pulmonary disease (COPD) is a major cause of mortality worldwide, and pulmonary epithelial cell apoptosis is regarded as one of the most important factors in its pathogenesis. Here, we examined the molecular mechanisms of apoptosis caused by cigarette smoke (CS). In the normal bronchial epithelium cell line BEAS-2B, a CS extract markedly induced apoptosis together with transient early growth response 1 (EGR1) protein expression, which is activated over time via the aryl hydrocarbon receptor (AHR). The CS extract-induced apoptosis decreased cell count of BEAS-2B cells and was significantly reversed by knockdown of either EGR1 or AHR. In vivo, the CS extract caused alveolar wall destruction, mimicking COPD, 1 week after intrathoracic injection. Bronchoalveolar lavage fluid (BALF) from the CS extract-treated mice contained massive numbers of apoptotic epithelial cells. Furthermore, it was found that aminoanthracene induced EGR1 expression and cell apoptosis. By contrast, the AHR antagonist stemregenin 1 (SR1) restored apoptosis upon CS treatment. These results suggest that aryl hydrocarbons, such as aminoanthracene, induce EGR1 expression via the AHR, resulting in cell apoptosis and that this can be prevented by administration of an antagonist of AHR.

Cigarette smoke, but not novel tobacco vapor products, causes epigenetic disruption and cell apoptosis.

Heat-Not-Burn (HNB) products, generating vapor without combusting tobacco leaves, have been developed with the expectation that the number and quantity of chemicals in the vapor of these products would be reduced compared with the smoke from conventional combustible cigarettes. However, whether the lower chemical levels correlate with lower toxicity remains to be determined. Here we examined differences in the biological effects of conventional cigarette smoke (CS) and two HNB products, Ploom TECH and Ploom TECH+, using the cultured cancer cell line A549 and the normal bronchial epithelium cell line BEAS-2B. The conventional CS 3R4F extract (0.5%) markedly decreased cell proliferation of both A549 and BEAS-2B cells; however, 0.5% extracts of these commercially available HNB products did not affect cell growth. To determine the cause of decreased cell proliferation, a TUNEL assay was performed, and the results indicated that apoptosis had occurred in both A549 and BEAS-2B cells at 24 h after exposure to 3R4F. To further explore the effect of CS on epigenetics, we performed western blotting to detect histone H2A phosphorylation, which is known to affect transcriptional regulation. Only the 3R4F extract decreased histone H2A phosphorylation in both A549 and BEAS-2B cells. Next, we examined alterations in gene expression after treatment of A549 cells with Ploom TECH, Ploom TECH+, or 3R4F extracts. It was found that 339, 107, and 103 genes were upregulated more than 2 fold in A549 cells treated with 3R4F, Ploom TECH, or Ploom TECH + extracts, respectively. Among the 339 genes that were upregulated in response to 3R4F, we focused on EGR1, FOS, and FOSB, since they were upregulated more than 100 fold, which was confirmed using RT-qPCR. These results suggest that CS, but not HNB products, cause epigenetic disruption and cell apoptosis, possibly by elevating transcription of genes such as EGR1.

Defects in centromeric/pericentromeric histone H2A T120 phosphorylation by hBUB1 cause chromosome missegregation producing multinucleated cells.

Histone H2A phosphorylation plays a role both in chromatin condensation during mitosis and in transcriptional activation during the G1/S transition. Bub1 and NHK1/VRK1 have been identified as histone H2A kinases. However, little is known about the importance of histone H2A phosphorylation in chromosome segregation. Here, we expressed recombinant hBUB1 and confirmed that it phosphorylates histone H2A T120 in the in vitro-assembled nucleosome. Knockdown (KD) of BUB1 decreases bulk H2A T120 phosphorylation in HeLa cells, whereas hBUB1 is upregulated during mitosis, which corresponds with H2A T120 phosphorylation. ChIP-qPCR of the DXZ1 centromeric and γ-ALR pericentromeric region showed that BUB1 localizes to this region and increases local H2A T120 phosphorylation during M phase. BUB1 KD did not induce apoptosis but increased the M phase cell population, as detected by flow cytometry. BUB1 KD also caused an abnormal metaphase and telophase, resulting in multinucleated cells and impaired cancer cell growth both in vitro and in vivo. Over-expression of the histone H2A T120D or T120E mutations, which mimic phosphorylated threonine, decreased the number of multinucleated cells caused by BUB1 KD. These results strengthen the apparent importance of BUB1-mediated H2A T120 phosphorylation in normal mitosis.

A case of an elderly man who required repeated repair of a ventricular septal defect and tricuspid rupture after blunt chest trauma.

Several cases of traumatic ventricular septal defect (VSD) have been reported. However, traumatic VSD complicated by tricuspid rupture is rare. We report a case of traumatic VSD with tricuspid rupture who required repeated repair of both conditions. A 69-year-old man was transferred to our hospital for emergent surgical repair of traumatic VSD and tricuspid rupture. Although emergent repair was performed, a new left-to-right shunt and moderate tricuspid regurgitation appeared during his postoperative course. A reoperation was performed 4 months after the first operation. The borders of the defect were very fibrotic and strong compared with those in the first operation. Surgical treatment of traumatic VSD should be postponed in hemodynamically stable patients. When emergent repair is performed, careful follow-up is necessary to diagnose new VSD.

SYNTAX Score and Long-Term Outcomes: The BARI-2D Trial.

BACKGROUND: The extent of coronary disease affects clinical outcomes and may predict the effectiveness of coronary revascularization with either coronary artery bypass graft (CABG) surgery or percutaneous coronary intervention (PCI). The SYNTAX (Synergy Between Percutaneous Coronary Intervention With Taxus and Cardiac Surgery) score quantifies the extent of coronary disease. OBJECTIVES: This study sought to determine whether SYNTAX scores predicted outcomes and the effectiveness of coronary revascularization compared with medical therapy in the BARI-2D (Bypass Angioplasty Revascularization Investigation 2 Diabetes) trial. METHODS: Baseline SYNTAX scores were retrospectively calculated for BARI-2D patients without prior revascularization (N = 1,550) by angiographic laboratory investigators masked to patient characteristics and outcomes. The primary outcome was major cardiovascular events (a composite of death, myocardial infarction, and stroke) over 5 years. RESULTS: A mid/high SYNTAX score (≥23) was associated with a higher risk of major cardiovascular events (hazard ratio: 1.36, confidence interval: 1.07 to 1.75, p = 0.01). Patients in the CABG stratum had significantly higher SYNTAX scores: 36% had mid/high SYNTAX scores compared with 13% in the PCI stratum (p < 0.001). Among patients with low SYNTAX scores (≤22), major cardiovascular events did not differ significantly between revascularization and medical therapy, either in the CABG stratum (26.1% vs. 29.9%, p = 0.41) or in the PCI stratum (17.8% vs. 19.2%, p = 0.84). Among patients with mid/high SYNTAX scores, however, major cardiovascular events were lower after revascularization than with medical therapy in the CABG stratum (15.3% vs. 30.3%, p = 0.02), but not in the PCI stratum (35.6% vs. 26.5%, p = 0.12). CONCLUSIONS: Among patients with diabetes and stable ischemic heart disease, higher SYNTAX scores predict higher rates of major cardiovascular events and were associated with more favorable outcomes of revascularization compared with medical therapy among patients suitable for CABG. (Bypass Angioplasty Revascularization Investigation in Type 2 Diabetes; NCT00006305).

Acute stent recoil and optimal balloon inflation strategy: an experimental study using real-time optical coherence tomography.

AIMS: Our aim was to evaluate stent expansion and acute recoil at deployment and post-dilatation, and the impact of post-dilatation strategies on final stent dimensions. METHODS AND RESULTS: Optical coherence tomography (OCT) was performed on eight bare metal platforms of drug-eluting stents (3.0 mm diameter, n=6 for each) during and after balloon inflation in a silicone mock vessel. After nominal-pressure deployment, a single long (30 sec) vs. multiple short (10 sec x3) post-dilatations were performed using a non-compliant balloon (3.25 mm, 20 atm). Stent areas during deployment with original delivery systems were smaller in stainless steel stents than in cobalt-chromium and platinum-chromium stents (p<0.001), whereas subsequent acute recoil was comparable among the three materials. At post-dilatation, acute recoil was greater in cobalt-chromium and platinum-chromium stents than in stainless steel stents (p<0.001), resulting in smaller final stent areas in cobalt-chromium and platinum-chromium stents than in stainless steel stents (p<0.001). In comparison between conventional and latest-generation cobalt-chromium stents, stent areas were not significantly different after both deployment and post-dilatation. With multiple short post-dilatations, acute recoil was significantly improved from first to third short inflation (p<0.001), achieving larger final area than a single long inflation, despite stent materials/designs (p<0.001). CONCLUSIONS: Real-time OCT revealed significant acute recoil in all stent types. Both stent materials/designs and post-dilatation strategies showed a significant impact on final stent expansion.

Bone Generation Profiling Around Photofunctionalized Titanium Mesh.

PURPOSE: The aim of this study was to evaluate whether photofunctionalization of titanium mesh enhances its osteoconductive capability. MATERIALS AND METHODS: The titanium mesh (0.2 mm thickness) used in this study was made of commercially pure grade-2 titanium and had hexagonal apertures (2 mm width). Photofunctionalization was performed by treating titanium mesh with UV light for 12 minutes using a photo device immediately before use. Untreated or photofunctionalized titanium mesh was placed into rat femurs, and bone generation around titanium mesh was profiled using three-dimensional (3D) microcomputed tomography (micro-CT). A set of in vitro experiments was conducted using bone marrow-derived osteoblasts. RESULTS: Photofunctionalized titanium mesh surfaces were characterized by the regenerated hydrophilicity and significantly reduced surface carbon. Bone generation profiling at week 3 of healing showed that the hexagonal apertures in photofunctionalized mesh were 95% filled, but they were only 57% filled in untreated mesh, particularly with the center zone remaining as a gap. Bone profiling in slices parallel to the titanium surface showed that photofunctionalized titanium mesh achieved 90% bone occupancy 0 to 400 µm from the surface, compared with only 35% for untreated mesh. Bone occupancy remained as high as 55% 800 to 1,200 µm from photofunctionalized titanium mesh surfaces, compared with less than 20% for untreated mesh. In vitro, photofunctionalized titanium mesh expedited and enhanced attachment and spread of osteoblasts, and increased ALP activity and the rate of mineralization. CONCLUSION: This study may provide novel and advanced metrics describing the osteoconductive property of photofunctionalized titanium mesh. Specifically, photofunctionalization not only increased the breadth, but also the 3D range, of osteoconductivity of titanium mesh, enabling space-filling and far-reaching osteoconductivity. Further translational and clinical studies are warranted to establish photofunctionalized titanium mesh as a novel clinical tool for better bone regeneration and augmentation.

Effect of UV Photofunctionalization on Biologic and Anchoring Capability of Orthodontic Miniscrews.

PURPOSE: Treatment of titanium with UV light immediately before use, or photofunctionalization, is gaining traction as a simple method to improve the biologic capability and clinical performance of dental implants. The objective of this study was to examine the effect of photofunctionalization on the biologic capability and mechanical anchorage of orthodontic miniscrews. MATERIALS AND METHODS: Untreated and photofunctionalized Ti-6Al-4V orthodontic miniscrews were placed into rat femurs. Photofunctionalization was performed by treating miniscrews with UV light for 12 minutes using a photo device immediately before placement. After 3 weeks of healing, miniscrews were pushed laterally to measure the resistance against the tipping force. The miniscrews were also evaluated for morphology and chemistry of tissue formed around them using scanning electron microscopy and energy dispersive spectroscopy. Rat bone marrow-derived osteoblasts were cultured on Ti-6Al-4V disks with and without photofunctionalization. The number of osteoblasts attached to the disks and the behaviors, alkaline phosphatase activity, and mineralization capability of osteoblasts were evaluated. RESULTS: Photofunctionalization converted both disk and screw surfaces from hydrophobic to superhydrophilic. In vivo biomechanical testing showed that the displacement of untreated screws was 1.5 to 1.7 times greater than that of photofunctionalized screws when subjected to lateral tipping force. Robust bone formation was observed around photofunctionalized miniscrews with strong elemental peaks of calcium and phosphorus, whereas the tissue around untreated miniscrews appeared thin and showed no clear peak of calcium. The attachment, initial spreading, adhesion, and expression of functional phenotypes of osteoblasts were significantly increased on photofunctionalized Ti-6Al-4V disks. CONCLUSION: These in vivo and in vitro results comprehensively and consistently demonstrate that photofunctionalization increases the bioactivity of Ti-6Al-4V and improves the anchoring capability of orthodontic miniscrews.

Biocompatibility of 4-META/MMA-TBB resin used as a dental luting agent.

STATEMENT OF PROBLEM: The bonding and biological properties of currently used luting/cementing materials need to be improved. 4-Acryloyloxyethyl trimellitate anhydride/methyl methacrylate-tri-n-butylborane (4-META/MMA-TBB) resin is primarily used for splinting mobile teeth or treating fractured teeth. It undergoes moisture-resistant polymerization and bonds strongly to dentin and metals. PURPOSE: The purpose of this in vitro study was to compare the biological and biochemical properties META/MMA-TBB resin with those of conventional polymethyl methacrylate (PMMA)-MMA resin and other currently used luting materials in order to determine whether it may be a viable dental luting agent. MATERIAL AND METHODS: The degree of polymerization of 4-META/MMA-TBB resin, PMMA-MMA autopolymerizing resin, 10-methacryloyloxydecyl dihydrogen phosphate-dimethacrylate (MDP-DMA) adhesive resin, and a glass ionomer cement was measured by Fourier-transformed infrared spectroscopy. Free radical production during setting was evaluated by electron spin resonance (ESR) spectroscopy. Rat dental pulp cells cultured on these materials were examined for cell viability, attachment, proliferation, and functional phenotype. RESULTS: The degree of polymerization of 4-META/MMA-TBB resin was 82% thirty minutes after preparation, compared to 66% for PMMA-MMA autopolymerizing resin. ESR spectroscopy revealed free radical production from 4-META/MMA-TBB resin and glass ionomer cement was equivalent 24 hours after preparation, with no spike in radical generation observed. In contrast, free radical production from PMMA-MMA and MDP-DMA adhesive resins was rapid and sustained and 10 to 20 times greater than that from 4-META/MMA-TBB. The percentage of viable dental pulp cells 24 hours after seeding was considerably higher on MDP-DMA and 4-META/MMA-TBB resin than on glass ionomer cement. Cell number, proliferation, and alkaline phosphatase activity were highest on 4-META/MMA-TBB resin and lowest on the glass ionomer cement. CONCLUSIONS: 4-META/MMA-TBB resin is at least as biocompatible, and perhaps even more biocompatible, than other current luting materials, with fast, favorable, and nontoxic polymerization properties. Further in vivo and human studies of 4-META/MMA-TBB resin as a dental luting agent are warranted.

Xylan deposition and lignification in the multi-layered cell walls of phloem fibres in Mallotus japonicus (Euphorbiaceae).

Phloem fibres in Mallotus japonicus Müll. Arg. were found to have a multi-layered structure that is S1 + S2 + n(G + L), where a non-lignified gelatinous layer (G) and a lignified layer (L) are formed alternately and n indicates the number of repetitions of these two layers. The aim of this study was to determine the process of xylan deposition and lignification in the multi-layered cell walls of phloem fibres. The formation process of the multi-layered structure of secondary phloem fibres was examined by light microscopy, ultraviolet microscopy and transmission electron microscopy. The distribution of glucuronoxylan was examined by immunoelectron microscopy. The activity of peroxidase was also determined using metal-enhanced diaminobenzidine substrates. Immunolabelling of glucuronoxylan occurred in lignified cell wall layers, except in the compound middle lamella (CML), i.e., the S1, S2 and L layers but not the G layers. Change in immunolabelling density suggests that xylan deposition in these lignified layers occurs appositionally, i.e., xylan is deposited into the lignified layers directly and not by a penetrative mechanism, and deposition does not occur after the layers are fully deposited. Peroxidase activity was found in CML including cell corners during S2 layer formation, then in developing G layers during G layer formation. Peroxidase activity was also found in the thin L layers that formed recently and was not found in the L layers already present. Xylan labelling was not found in the thin L layers that formed recently but did occur in L layers that developed earlier. Lignification of the S1 and S2 layers continued during the formation of the G layers, whereas in the L layers it finished just after deposition of the L layer.

Early-stage osseointegration capability of a submicrofeatured titanium surface created by microroughening and anodic oxidation.

OBJECTIVE: The role of nanoscale/submicron morphological features in the process of osseointegration is largely unknown. This study reports the creation of a unique submicrofeatured titanium surface by a combination of anodic oxidation and sandblasting and determines how the addition of this submicrofeature to a microroughened surface affects the early-stage process of osseointegration. MATERIALS AND METHODS: Nonmicroroughened implants were prepared by machining Ti-6Al-4V alloy in a cylindrical form (1 mm diameter and 2 mm long). Microroughened implants were prepared by sandblasting machined implants, while submicrofeatured implants were created by anodic oxidation of the sandblasted implants. Implants were placed into rat femurs and subjected to biomechanical, interfacial, and histological analyses at 1 and 2 weeks post-implantation (n = 6). RESULTS: The submicrotopography was characterized by 50-300 nm nodules and pits in addition to other submicron-level irregularities formed entirely within the sandblast-created microstructures. The biomechanical strength of osseointegration increased continuously from week 1 to 2 for the submicrofeatured implants but not for the microroughened implants. A significant increase in bone-implant contact and bone volume, as well as a reduction in soft tissue intervention, were commonly found for the microroughened surface and the submicrofeatured surface compared with the nonmicroroughened surface. However, there were no differences in these parameters between the microroughened surface and the submicrofeatured surface. An extensive area of bone tissue at the submicrofeatured implant interface was retained intact after biomechanical shear testing, while the microroughened implant-tissue interface showed a gap along the entire axis of the implant, leading to clear separation of the tissue during the shear procedure. CONCLUSIONS: This study demonstrates that a submicrofeatured titanium surface created by a combination of sandblasting and anodic oxidation enhances the strength of early-stage osseointegration, primarily because of the increased resistance of peri-implant bone tissue against external force rather than modulation of bone morphogenesis.

Anatomy and lignin distribution in reaction phloem fibres of several Japanese hardwoods.

BACKGROUND AND AIMS: Although tension wood formation and the structure of gelatinous fibres (G-fibres) have been widely investigated, studies of the influence of the reaction phenomenon on phloem fibres have been few and incomplete in comparison with those of xylem wood fibres. This study was undertaken to clarify the influence of stem inclination on phloem fibres using several Japanese hardwood species that produce different G-fibre types in tension wood. METHODS: Eight hardwood species were inclined at 30-45° at the beginning of April. Specimens were collected in July and December. The cell-wall structure and lignin distribution of phloem fibres on both the tension and opposite sides were compared by light microscopy, ultraviolet microscopy, confocal laser scanning microscopy after staining with acriflavine, and transmission electron microscopy after staining with potassium permanganate. KEY RESULTS: Three types of changes were found in tension-side phloem fibres: (1) increases in the proportion of the syringyl unit in lignin in the S(1) and S(2) layers and compound middle lamella (Cercidiphyllum japonicum), (2) formation of unlignified gelatinous layers (Melia azedarach and Acer rufinerve) and (3) increases in the number of layers (n) in the multi-layered structure of S(1) + S(2) + n (G + L) (Mallotus japonicus). Other species showed no obvious change in cell-wall structure or lignin distribution. CONCLUSIONS: Phloem fibres of the tree species examined in our study showed three types of changes in lignin distribution and cell-wall structure. The reaction phenomenon may vary with tree species and may not be closely related to G-fibre type in tension wood.

Bone integration capability of nanopolymorphic crystalline hydroxyapatite coated on titanium implants.

The mechanism by which hydroxyapatite (HA)-coated titanium promotes bone-implant integration is largely unknown. Furthermore, refining the fabrication of nano-structured HA to the level applicable to the mass production process for titanium implants is challenging. This study reports successful creation of nanopolymorphic crystalline HA on microroughened titanium surfaces using a combination of flame spray and low-temperature calcination and tests its biological capability to enhance bone-implant integration. Sandblasted microroughened titanium implants and sandblasted + HA-coated titanium implants were subjected to biomechanical and histomorphometric analyses in a rat model. The HA was 55% crystallized and consisted of nanoscale needle-like architectures developed in various diameters, lengths, and orientations, which resulted in a 70% increase in surface area compared to noncoated microroughened surfaces. The HA was free from impurity contaminants, with a calcium/phosphorus ratio of 1.66 being equivalent to that of stoichiometric HA. As compared to microroughened implants, HA-coated implants increased the strength of bone-implant integration consistently at both early and late stages of healing. HA-coated implants showed an increased percentage of bone-implant contact and bone volume within 50 µm proximity of the implant surface, as well as a remarkably reduced percentage of soft tissue intervention between bone and the implant surface. In contrast, bone volume outside the 50 µm border was lower around HA-coated implants. Thus, this study demonstrated that the addition of pure nanopolymorphic crystalline HA to microroughened titanium not only accelerates but also enhances the level of bone-implant integration and identified the specific tissue morphogenesis parameters modulated by HA coating. In particular, the nanocrystalline HA was proven to be drastic in increasing osteoconductivity and inhibiting soft tissue infiltration, but the effect was limited to the immediate microenvironment surrounding the implant.

Bone integration capability of alkali- and heat-treated nanobimorphic Ti-15Mo-5Zr-3Al.

The role of nanofeatured titanium surfaces in a number of aspects of in vivo bone-implant integration, and, in particular, their potential advantages over microfeatured titanium surfaces, as well as their specific contribution to osteoconductivity, is largely unknown. This study reports the creation of a unique nanobimorphic titanium surface comprised of nanotrabecular and nanotuft-like structures and determines how the addition of this nanofeature to a microroughened surface affects bone-implant integration. Machined surfaces without microroughness, sandblasted microroughened surfaces, and micro-nano hybrid surfaces created by sandblasting and alkali and heat treatment of Ti-15Mo-5Zr-3Al alloy were subjected to biomechanical, interfacial and histological analyses in a rat model. The presence of microroughness enabled accelerated establishment of biomechanical implant fixation in the early stages of healing compared to the non-microroughened surfaces; however, it did not increase the implant fixation at the late stages of healing. The addition of nanobimorphic features to the microroughened surfaces further increased the implant fixation by as much as 60-100% over the healing time. Bone area within 50 µm of the implant surface, but not beyond this distance, was significantly increased by the presence of nanobimorphic features. Although the percentage of bone-implant contact was also significantly increased by the addition of nanobimorphic features, the greatest improvement was found in the soft tissue intervention between the bone and the implant, which was reduced from >30% to <5%. Mineralized tissue densely deposited with calcium-binding globular proteins was observed in an extensive area of nanobimorphic surfaces after biomechanical testing. This study clearly demonstrates the nanofeature-enhanced osteoconductivity of titanium by an alkali- and heat-treated nanobimorphic surface compared to that by microfeatured surfaces, which results not only in an acceleration but also an improvement of bone-implant integration. The identified biological parameters that successfully detect the advantages of nanofeatures over microfeatures will be useful in evaluating new implant surfaces in future studies.

The fission yeast protein Ker1p is an ortholog of RNA polymerase I subunit A14 in Saccharomyces cerevisiae and is required for stable association of Rrn3p and RPA21 in RNA polymerase I.

A heterodimer formed by the A14 and A43 subunits of RNA polymerase (pol) I in Saccharomyces cerevisiae is proposed to correspond to the Rpb4/Rpb7 and C17/C25 heterodimers in pol II and pol III, respectively, and to play a role(s) in the recruitment of pol I to the promoter. However, the question of whether the A14/A43 heterodimer is conserved in eukaryotes other than S. cerevisiae remains unanswered, although both Rpb4/Rpb7 and C17/C25 are conserved from yeast to human. To address this question, we have isolated a Schizosaccharomyces pombe gene named ker1+ using a yeast two-hybrid system, including rpa21+, which encodes an ortholog of A43, as bait. Although no homolog of A14 has previously been found in the S. pombe genome, functional characterization of Ker1p and alignment of Ker1p and A14 showed that Ker1p is an ortholog of A14. Disruption of ker1+ resulted in temperature-sensitive growth, and the temperature-sensitive deficit of ker1delta was suppressed by overexpression of either rpa21+ or rrn3+, which encodes the rDNA transcription factor Rrn3p, suggesting that Ker1p is involved in stabilizing the association of RPA21 and Rrn3p in pol I. We also found that Ker1p dissociated from pol I in post-log-phase cells, suggesting that Ker1p is involved in growth-dependent regulation of rDNA transcription.

The fission yeast RPA51 is a functional homolog of the budding yeast A49 subunit of RNA polymerase I and required for maximizing transcription of ribosomal DNA.

Saccharomyces cerevisiae A49 and mouse PAF53 are subunits specific to RNA polymerase I (Pol I) in eukaryotes. It has been known that Pol I without A49 or PAF53 maintains non-specific transcription activities but a molecular role(s) of A49 (and PAF53) remains totally unknown. We studied the fission yeast gene encoding a protein of 415 amino acids exhibiting 30% and 19% identities to A49 and PAF53, respectively. We designate the corresponding protein RPA51 and gene encoding it rpa51+ since the gene encodes a Pol I subunit and an apparent molecular mass of the protein is 51 kDa. rpa51+ is required for cell growth at lower but not at higher temperatures and is able to complement S. cerevisiae rpa49Delta mutation, indicating that RPA51 is a functionally-conserved subunit of Pol I between the budding yeast and the fission yeast. Deletion analysis of rpa51+ shows that only two-thirds of the C-terminal region are required for the function. Transcripts analysis in vivo and in vitro shows that RPA51 plays a general role for maximizing transcription of rDNA whereas it is dispensable for non-specific transcription. We also found that RPA51 associates significantly with Pol I in the stationary phase, suggesting that Pol I inactivation in the stationary phase of yeast does not result from the RPA51 dissociation.

The fission yeast RPA21 subunit of RNA polymerase I: an evolutionarily conserved subunit interacting with ribosomal DNA (rDNA) transcription factor Rrn3p for recruitment to rDNA promoter.

Recruitment of RNA polymerases to the cognate promoter is a key step for the transcription initiation of specific genes in eukaryotes. Recently, RNA polymerase I (pol I) of Saccharomyces cerevisiae was shown to be recruited to the rDNA promoter via interaction between Rrn3p, a conserved transcription factor for rDNA, and A43, a subunit specific to pol I. The question of whether a similar interaction for pol I recruitment is conserved in other eukaryotes remains to be answered. We show here that Schizosaccharomyces pombe rpa21(+) encodes a protein of apparent molecular mass 21 kD which shows 36% identity to the A43 subunit of pol I in S. cerevisiae, and that rpa21(+) is essential for cell growth. To gain further insight into the functions of RPA21, we isolated a total of 22 temperature-sensitive (ts) mutants of rpa21(+) and found that most of the substitutions causing the ts phenotype are clustered in the N-terminal half of RPA21. The ts mutants showed a markedly reduced amount of primary transcripts of rDNA immediately after temperature shift-up. Over-expression of S. pombe rrn3(+) in the ts mutants suppressed the growth defect in an allele-specific manner. Therefore, we conclude that S. pombe RPA21 plays a functional role similar to that of A43 in S. cerevisiae and that the mechanism of recruitment of pol I to the rDNA promoter by the interaction of a specific pol I subunit with Rrn3p is evolutionarily conserved.