Deletion of the RNA polymerase subunit RPB4 acts as a global, not stress-specific, shut-off switch for RNA polymerase II transcription at high temperatures.

We used whole genome expression analysis to investigate the changes in the mRNA profile in cells lacking the Saccharomyces cerevisiae RNA polymerase II subunit RPB4 (Delta RPB4). Our results indicated that an essentially complete shutdown of transcription occurs upon temperature shift of this conditionally lethal mutant; 98% of mRNA transcript levels decrease at least 2-fold, 96% at least 4-fold. This data was supported by in vivo experiments that revealed a rapid and greater than 5-fold decline in steady state poly(A) RNA levels after the temperature shift. Expression of several individual genes, measured by Northern analysis, was also consistent with the whole genome expression profile. Finally we demonstrated that the loss of RNA polymerase II activity causes secondary effects on RNA polymerase I, but not RNA polymerase III, transcription. The transcription phenotype of the Delta RPB4 mutant closely mirrors that of the temperature-sensitive rpb1-1 mutant frequently implemented as a tool to inactivate the RNA polymerase II in vivo. Therefore, the Delta RPB4 mutant can be used to easily design strains that enable the study of distinct post-transcriptional cellular processes in the absence of RNA polymerase II transcription.

T cell apoptosis causes peripheral T cell depletion in mice transgenic for the HIV-1 vpr gene.

Vpr, an accessory protein of HIV, is known to affect viral replication as well as cell growth, differentiation, and apoptosis in vitro. To investigate its pathogenicity in vivo, we have produced mice transgenic for the HIV-1 vpr gene with the CD4 enhancer/promoter. Interestingly, apoptotic death of T lymphocytes was enhanced in those mice, causing marked reduction of T cells in lymphatic organs and peripheral blood. Involvement of Bcl-x, Bax, and Caspase-1, but not of the Fas-Fas ligand system, was suggested in the apoptotic processes. These observations suggest that Vpr is involved in the pathogenesis of T cell depletion in HIV-infected people.

85Kr measurement system for continuous monitoring at the Meteorological Research Institute, Japan.

A 85Kr measurement system for continuous monitoring based principally on the BfS-IAR method (activity measurement of 85Kr by gas counting coupled with gas chromatographic separation, using pure CH4 as carrier and Counting gas) was implemented for the first time in Japan. In this paper, a detailed description of the system and procedures is given and the inter-comparison results of our system with the BfS-IAR system are presented. A consistent temporal concentration change with high accuracy and consistency of the respective data with the BfS-IAR data (maximum difference of 5%) were achieved with the Meteorological Research Institute (MRI) system, which shows that the system is valid and reliable for the purpose of background monitoring for 85Kr in air. Also, the 85Kr monitoring record at the MRI during 1995-2001 is described. The record distinctively shows the Northern Hemispheric background 85Kr concentrations at the mid-latitude and the elevated concentrations affected by the operation of a nuclear fuel reprocessing plant in Tokai-mura, Ibaraki. Japan.

Multiple mechanisms of suppression circumvent transcription defects in an RNA polymerase mutant.

Using a high-copy-number suppressor screen to obtain clues about the role of the yeast RNA polymerase II subunit RPB4 in transcription, we identified three suppressors of the temperature sensitivity resulting from deletion of the RPB4 gene (DeltaRPB4). One suppressor is Sro9p, a protein related to La protein, another is the nucleosporin Nsp1p, and the third is the RNA polymerase II subunit RPB7. Suppression by RPB7 was anticipated since its interaction with RPB4 is well established both in vitro and in vivo. We examined the effect of overexpression of each suppressor gene on transcription. Interestingly, suppression of the temperature-sensitive phenotype correlates with the correction of a characteristic transcription defect of this mutant: each suppressor restored the level of promoter-specific, basal transcription to wild-type levels. Examination of the effects of the suppressors on other in vivo transcription aberrations in DeltaRPB4 cells revealed significant amelioration of defects in certain inducible genes in Sro9p and RPB7, but not in Nsp1p, suppressor cells. Analysis of mRNA levels demonstrated that overexpression of each of the three suppressors minimally doubled the mRNA levels during stationary phase. However, the elevated mRNA levels in Sro9p suppressor cells appear to result from a combination of enhanced transcription and message stability. Taken together, these results demonstrate that these three proteins influence transcription and implicate Sro9p in both transcription and posttranscription events.

Low level 137Cs measurements in deep seawater samples

The ammonium phosphomolybdate (AMP) procedure for low level 137Cs measurements in deep seawater samples was examined, as a marked decrease in the recovery of AMP has been observed over several years. An improved AMP procedure is proposed: adjust the pH to 1.6-2.0, next add 0.26 g CsCl to form an insoluble compound, then add 4 g AMP to every 20-100 l seawater sample. Stir at a rate of 25 l air/min for 1 h, then recover the AMP/Cs compound after 6-24 h. The weight yield of AMP/Cs compound was about 98% of AMP for 20 l samples and the radiochemical 137Cs yield was 96-100%. The absolute efficiency of the HPGe coaxial well detector was 16% for 137Cs at 662 keV for 4 g AMP/Cs compound. Using the improved procedure, 137Cs activities in 20-100 l samples from sea surface to near bottom in the western North Pacific were measured. The 137Cs concentrations in deep water samples were lower than 0.1 Bq m(-3) in 1997 and 1998. This procedure allows a wide range of applications of 137Cs as a transient tracer for oceanographic purposes, as high resolution water profiles are obtained due to the smaller volume of samples needed.

Anthropogenic radionuclides in seawater of the Far Eastern Seas.

Large quantities of radioactive wastes have been dumped in the Far Eastern Seas by the former Soviet Union and the Russian Federation, and small amounts of radioactive wastes have been dumped by Japan and the Republic of Korea. In order to investigate the concentrations of anthropogenic radionuclides in the nine dumping areas, a second expedition was conducted in 1995 by Japan, the Republic of Korea, the Russian Federation and IAEA, following the first expedition in 1994. The results show that 137Cs, 90Sr and 239 + 240Pu concentrations in surface and bottom waters at dumping areas do not significantly differ from the values observed in background areas, and from historical values. There is no clear effect of possible contamination due to radioactive waste dumping. The concentrations and water column inventories of 137Cs, 90Sr and 239 + 240Pu in the Far Eastern seas are controlled by physical oceanic processes such as horizontal transport and biogeochemical processes such as scavenging.

Air concentration of radiocaesium in Tsukuba, Japan following the release from the Tokai waste treatment plant: comparisons of observations with predictions.

On March 11, 1997 a fire and explosion accident occurred at the bituminization facility of the Power Reactor and Nuclear Fuel Development, Tokai, Japan. As a result of this accident, 134,137Cs was detected in an air filter sample collected at the Meteorological Research Institute, Tsukuba during March 10 to 12. The 134,137Cs air concentration was about 100 and 10 muBq m-3, respectively. This result suggests that there was little radiation exposure of the residents in the area. The average 137Cs air concentration during this period was about two orders of magnitude higher than "baseline" air (sub-muBq m-3) during February to April, 1997, measured by ultra-low background gamma-spectrometry. By a simple calculation using a Gaussian plume model with the measured data, we estimated the minimum emission of the radioactivity by the PNC accident to be in the range 60 MBq to around 600 MBq. The meteorological condition during the week of the accident are also described.

RNA polymerase subunit RPB5 plays a role in transcriptional activation.

A mutation in RPB5 (rpb5-9), an essential RNA polymerase subunit assembled into RNA polymerases I, II, and III, revealed a role for this subunit in transcriptional activation. Activation by GAL4-VP16 was impaired upon in vitro transcription with mutant whole-cell extracts. In vivo experiments using inducible reporter plasmids and Northern analysis support the in vitro data and demonstrate that RPB5 influences activation at some, but not all, promoters. Remarkably, this mutation maps to a conserved region of human RPB5 implicated by others to play a role in activation. Chimeric human-yeast RPB5 containing this conserved region now can function in place of its yeast counterpart. The defects noted with rpb5-9 are similar to those seen in truncation mutants of the RPB1-carboxyl terminal domain (CTD). We demonstrate that RPB5 and the RPB1-CTD have overlapping roles in activation because the double mutant is synthetically lethal and has exacerbated activation defects at the GAL1/10 promoter. These studies demonstrate that there are multiple activation targets in RNA polymerase II and that RPB5 and the CTD have similar roles in activation.

Mapping of Rpb3 and Rpb5 contact sites on two large subunits, Rpb1 and Rpb2, of the RNA polymerase II from fission yeast.

[Rpb1 and Rpb2] Mapping of the contact sites on two large subunits of the fission yeast Schizosaccharomyces pombe RNA polymerase II with two small subunits, Rpb3 and Rpb5, was carried out using the two-hybrid screening system in the budding yeast Saccharomyces cerevisiae. Rpb5 was found to interact with any fragment of Rpb1 that contained the region H, which is conserved among the subunit 1 homologues of all RNA polymerases, including the beta' subunit of prokaryotic RNA polymerases. In agreement with the fact that Rpb5 is shared among all three forms of eukaryotic RNA polymerases, the region H of RNA polymerase I subunit 1 (Rpa190) was also found to interact with Rpb5. On the other hand, two-hybrid screening of Rpb2 fragments from RNA polymerase II indicated the presence of an Rpb3 contact site in the region H which is conserved among the subunit 2 homologues of all RNA polymerases, including the beta subunit of prokaryotic RNA polymerases. Possible functions of the regions H in the subunits 1 and 2 are discussed.

Central island tongue flap.

Pedicled tongue flaps have proved to be an effective method of repairing defects due to tissue loss in the oral cavity. Their central position, mobility, and excellent blood supply make their use feasible in a variety of sites. This paper describes the use and applications of central island tongue flaps to reconstruct defects of anterior floor of the mouth. This procedure was conducted at the Department of Oral and Maxillofacial Surgery, Medical University Hanover, introducing an improved surgical method and presenting the actual operation performed in our department. This method is considered superior for resurfacing the anterior floor of mouth defects because it is easy to perform and results in recovery of function and cosmetics.

Subunit composition of RNA polymerase II from the fission yeast Schizosaccharomyces pombe.

The subunit composition of RNA polymerase II (polII) was compared between the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe. For this purpose, we partially purified the enzyme from S. pombe. Judging from the co-elution profiles in column chromatographies of both the RNA polymerase activity and the two large subunit polypeptides (subunit 1 (prokaryotic beta' homologue) and subunit 2 (beta homologue)), the minimum number of S. pombe polII-associated polypeptides was estimated to be ten, less than the proposed subunit number of the S. cerevisiae enzyme. These ten putative subunits of S. pombe polII correspond to subunits 1, 2, 3, 5, 6, 7, 8, 10, 11 and 12 of the S. cerevisiae counterparts.

Molecular assembly of RNA polymerase II from the fission yeast Schizosaccharomyces pombe: subunit-subunit contact network involving Rpb5.

BACKGROUND: Eukaryotic RNA polymerase II is composed of more than 10 polypeptide chains. The minimum and essential subunits for RNA synthesis have not yet been identified. Toward this ultimate goal, we analysed the topological arrangement of the putative subunits. Here we report a subunit-subunit contact network involving subunit 5 of the fission yeast Schizosaccharomyces pombe RNA polymerase II. RESULTS: The rpb5+ gene encoding subunit 5 of RNA polymerase II was cloned from the fission yeast Schizosaccharomyces pombe. The polypeptide predicted from DNA sequence of the rpb5+ gene consists of 210 amino acids with a calculated molecular weight of 23914. The homology of the amino acid sequence is 55% and 43% with Saccharomyces cerevisiae RPB5 and human hRPB25, respectively. Far-Western blot analysis of S. pombe RNA polymerase II using 32P-labelled recombinant Rpb5 fused to glutathione S-transferase (GST) as a probe, indicated that Rpb5 binds strongly to membrane-immobilized Rpb1, Rpb2 and Rpb3 and weakly to Rpb5 and a 15-kDa subunit (Rpb8 or Rpb11). In agreement with this result, the 32P-labelled Rpb3 probe showed a strong binding signal against Rpb5 in addition to Rpb1 and Rpb2. The existence of Rpb5-Rpb3 contact was supported by detection of complexes formed between these two proteins synthesized in vitro using protein-immobilized beads. CONCLUSION: Rpb3 and Rpb5, the putative subunits of RNA polymerase II, associate each other to form binary complexes. These two subunits also bind to the two large subunits, Rpb1 and Rpb2, independently.

Stability and pharmacokinetic characteristics of oligonucleotides modified at terminal linkages in mice.

To construct the strategy for delivery systems that can control in vivo disposition of antisense oligonucleotides, we studied the stability and basic pharmacokinetic characteristics of oligonucleotides. Decathymidylic acid (T10), a model oligodeoxynucleotide, and its derivatives, 5'-biotin-T10) and 3'-methoxyethylamine 5'-biotin-T10 (3'M5'B-T10), containing phosphoroamidate modification at 3'- and/or 5'-terminal internucleoside linkages, were synthesized. In phosphate-buffered saline (PBS, pH 7.4) containing 10% mouse serum, unmodified T10 was degraded with a half-life of 45 minutes; the degradation half-lives of 5'B-T10 and 3'M5'B-T10 were 11 and 30 h, respectively. In mouse whole blood, 3'M5'B-T10 was relatively stable, and 45% remained intact after 1 h incubation. After intravenous injection of [3H]3'M5'B-T10 into mice at a dose of 1 mg/kg, the radioactivity was rapidly cleared from plasma with a half-life of 2 minutes and accumulated in the kidney, liver, and gallbladder. About 30% of the dose was excreted in the urine within 60 minutes. A much more rapid degradation of [3H]3'M5'B-T10 was observed in vivo than expected from in vitro experiments: more than 90% of the radioactivity in plasma was degradation product at 2 minutes after injection. These results suggested that enzymatic degradation occurred in some compartments in addition to the blood pool. The apparent urinary excretion clearance of [3H]3'M5'B-T10 was close to that of inulin, whereas the apparent hepatic uptake clearance was much greater than that of inulin and comparable to that of dextran sulfate, which is taken up by the liver by scavenger receptors for polyanions.(ABSTRACT TRUNCATED AT 250 WORDS)

Renal disposition characteristics of oligonucleotides modified at terminal linkages in the perfused rat kidney.

To clarify the renal disposition characteristics of oligonucleotides at the organ level, the renal handling of model end-capped oligonucleotides, 3'-methoxyethylamine 5'-biotin-decathymidylic acid containing phosphoramidate modifications at 3'- and 5'-terminal internucleoside linkages (T10) and its phosphorothioate (Ts10), were studied in the perfused rat kidney. In a single-pass indicator dilution experiment, venous outflow and urinary excretion patterns and tissue accumulation of radiolabeled oligonucleotides were evaluated under filtering or nonfiltering conditions. No significant binding to bovine serum albumin (BSA) in the perfusate was observed for T10, whereas more than 90% of Ts10 bound to BSA. The steady-state distribution volume of T10 calculated from the venous outflow pattern was larger than that of inulin, which corresponds to the extracellular volume of the kidney, whereas the distribution volume of Ts10 was larger than that of BSA (the intravascular volume). These results suggested their interaction with the vascular wall. Rapid urinary excretion was observed for T10, similar to inulin used as a marker of golmerular filtration rate. On the other hand, urinary excretion of Ts10 was greatly restricted due to its high binding ability (> 90%) to BSA in the perfusate. A significant amount of T10 and Ts10 was accumulated in the kidney (T10, 1.8% of injected dose; Ts10, 1.3%) compared with inulin (0.2%) and BSA (< 0.1%). The accumulation of these oligonucleotides was ascribed to both tubular reabsorption and uptake from the capillary side. In addition, the uptake of T10 from the capillary side was significantly inhibited by simultaneous injection of dextran sulfate, suggesting that the oligonucleotide was taken up as an anionic molecule. These findings will be useful information for the development of delivery systems for antisense oligonucleotides.

[Control of in vivo characteristics of gene and antisense DNA disposition].

In order to construct a strategy for control of in vivo disposition characteristics of gene and antisense DNA, in vivo stability and basic pharmacokinetic properties of DNA were investigated. A model gene, plasmid DNA (pCAT), and model oligonucleotide (thymidine 10-mer; T10) derivatives with phosphoroamidate substitution at the 3' and/or 5'-terminal internucleotide linkage, were rapidly degraded in vivo after intravenous injection into mice. The degradation rates were much faster than those observed in in vitro experiments using plasma and whole blood, suggesting that they underwent enzymatic degradation in other compartments than the blood pool. More than 70% of injected pCAT was taken up by the liver within 5 minutes, and the uptake clearance was almost identical to the plasma flow rate in the liver. On the other hand, T10 derivatives were rapidly excreted in the urine and taken up by the kidney and liver. The urinary excretion clearance was close to the glomerular filtration rate. In an attempt to control T10 disposition characteristics, the oligonucleotide was conjugated to a macromolecular carrier, carboxymethyl dextran (CMD). The T10-CMD conjugate exhibited increased in vivo stability and prolonged plasma circulation time. Thus, the present study has shown that macromolecular conjugation is a useful approach to improve in vivo disposition of antisense oligonucleotides.

Radioiodinated nordiazepam analog for in vivo assessment of benzodiazepine receptors by single photon emission tomography.

2'-Iodo-nordiazepam (2'-IND), a nordiazepam analog iodinated at the 2'-position of the C-5 phenyl ring, was synthesized and evaluated as a potential radiopharmaceutical for investigating brain benzodiazepine receptors by SPECT. [125I]2'-IND was synthesized by the halogen exchange reaction and purified by HPLC. In an in vitro competitive binding study using [3H]diazepam and rat cortical synaptosomol membranes, 2'-IND showed an almost equal affinity for benzodiazepine receptors as diazepam. In a saturation binding study using rat cortical synaptosomal membranes, 2'-IND displayed a Kd of 1.10 nM and a Bmax of 1.87 pmol/mg protein. Biodistribution and metabolism studies in mice showed that [125I]2'-IND exhibited rapid and high accumulation in the brain, and that the cerebral uptake and distribution of this compound occurred in the intact form. Furthermore, the administration of diazepam and flumazenil reduced cortical uptake by approx. 20%, suggesting that the uptake of 2'-IND occurred at least partly in association with benzodiazepine receptors.

A human skeleton from the Ohguruwa remains.

In 1941, the Ohguruwa remains were discovered at the Mizuho sports ground site in Nagoya. They date from about 3000 B.C., which is the early Jomon era. When the stadium was reconstructed in 1980, four human skeletons were found. Three of them, however, were in poor condition and moreover, were incomplete. However, the second skeleton was in good condition and could almost be reconstructed, and this skeleton (No. 2) was used for our study. It was found in the classic posture with arms and legs folded. Some pieces of a dog's skeleton were also discovered near the No. 2 skeleton's chest. This particular skeleton generally showed characteristics typical of the Jomon era. It had a stout structure and was judged to be a middle aged male because of the following features. There was considerable attrition of the occlusal surfaces on the remaining teeth. The teeth were worn flat, probably owing to the hard food and to their use as a tool. Both canines and first premolars of the upper and lower arches had been extracted in accordance with the custom of the time. Typical caries and localized periodontal breakdown were not observed, although there was horizontal alveolar bone loss, especially in the anterior regions. The mastoid process was extremely large and prominent. The lateral prominence of the mandible was developed. Analysis of lateral cephalogram revealed that the mandible was in the anterior position. The angles of SNA, SNB and ANB were 89.6 degrees, 89.2 degrees and 0.4 degrees, respectively. The skeletal pattern was definitive Class III. The adaptive changes in the teeth, their supporting tissues, temporomandibular joints and the related muscles--the harmonious masticatory system--were all estimated.

Long-run urban growth with agglomeration economies.

"It is widely recognized that agglomeration economies are a crucially important factor in explaining the existence and growth of urban areas, and therefore should be explicitly taken into consideration in long-run urban growth analysis. Once such economies are introduced, however, the urban economy tends to diverge from a steady state equilibrium and may 'explode' without limit. A possible way to solve this dilemma is shown. First, a simple urban growth model with production and factor migration functions in the presence of agglomeration economies is set up....Then, land is introduced to show that the availability of the third factor of production will make it more likely to achieve a steady growth equilibrium in the presence of agglomeration economies. Last, the model is generalized to include many factors of production."

Rural and urban population changes and the stages of economic development: a unified approach.

The long-term aspects of the process of economic development and urbanization are examined. A model is presented that shows the dynamics of economic development from the earliest to the more advanced stages. "The model is able to explain not only the occurrence of a downturn in the rural population after the initial phase of population growth both in rural and urban areas, but also the delayed occurrence of such a downturn in many present-day developing countries. The author then focuses [on] the later stages of economic development and explains two alternative courses of urbanization, namely, the reversal process and the continual-growth process, as special cases of the general model; which of the courses occurs depends on the value of the elasticity of urban agglomeration-economies."