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The progesterone receptor (PR) plays an important role in various physiological processes, especially in the female reproductive system, and abnormalities of PR function are associated with several diseases, including some types of cancer. Non-steroidal PR ligands are of interest as candidate drugs for treatment of PR-related diseases without the serious adverse effects that may be caused by steroidal ligands. For the development of non-steroidal PR ligands, both a hydrophobic backbone and a polar functional group corresponding to the 3-carbonyl group of progesterone, which interacts with Gln725 and Arg766 of the PR-ligand binding domain, are critically important. We previously showed that carborane is a useful hydrophobic pharmacophore for PR antagonists, and in this work, we introduced the pentafluorosulfanyl (SF5) group as a novel polar functional group of carborane-based non-steroidal PR antagonists. All the synthesized SF5-containing carborane derivatives exhibited PR-antagonistic activity at micromolar or submicromolar concentration. Among them, compounds 11 are potent progesterone antagonists with submicromolar IC50 values.

Thermal impedance, or thermal resistance, is an important parameter which has been used to evaluate the thermal performance of electronic packages and construct large scale thermal simulation models. The thermal impedance of an electronic package can be affected by almost all construction details, such as thermal properties of Epoxy Molding Compound (EMC), size and thickness of each die, substrate construction, layout and material properties of solder ball, etc. To get a full thermal impedance report, simulations in eight JEDEC environments need to be carried out. The whole simulation process can cost several hours, and it is hard to avoid mistakes when collecting numerous construction details and building complex models. To reduce the simulation cost and minimize the chance of error, a simple but accurate method has been developed. This method can generate a thermal impedance report within several minutes and the results are comparable with simulation outcomes. Finally, we also show how to improve thermal performance of memory packages by reducing thermal resistance and use this as a guideline for future thermal design of any electronic packages.

The progesterone receptor (PR) plays an important role in various physiological processes, especially in the female reproductive system, and abnormalities of PR function are associated with several diseases, including some types of cancer. Non-steroidal PR ligands are of interest as candidate drugs for treatment of PR-related diseases without the serious adverse effects that may be caused by steroidal ligands. For the development of non-steroidal PR ligands, both a hydrophobic backbone and a polar functional group corresponding to the 3-carbonyl group of progesterone, which interacts with Gln725 and Arg766 of the PR-ligand binding domain, are critically important. We previously showed that carborane is a useful hydrophobic pharmacophore for PR antagonists, and in this work, we introduced the pentafluorosulfanyl (SF

Progesterone receptor (PR) regulates various physiological processes, including the female reproductive system, and development of nonsteroidal PR antagonists is considered desirable for clinical application, as they are expected to have reduced side effects. We have synthesized a series of nonsteroidal PR antagonists using a 4-cyanophenyl- p -carborane core structure. Among them, compound 14d exhibited potent PR-antagonistic activity (IC 50 : 27 nM). It showed high binding affinity for PR, but did not bind to androgen receptor or estrogen receptor. This PR-selective antagonist may be a promising lead compound for clinically applicable progesterone receptor modulators.

The progesterone receptor (PR) plays important roles in multiple physiological processes, including female reproduction. Here, we report the synthesis of nonsteroidal PR antagonists containing a boron cluster as the hydrophobic core. We found that 1,7-diphenyl-meta-carborane was the preferred substructure among the three carborane isomers. Compound 39 was the most potent PR antagonist (IC50: 29 nM). Compound 41 also exhibited potent activity (IC50: 93 nM), and did not bind to androgen receptor, glucocorticoid receptor or mineralocorticoid receptor. These compounds may be useful for investigating potential clinical applications of PR modulators.

We have constructed the recombinant phage harboring the genes that code for enzymes for cysteine biosynthesis with acetylCoA regeneration system by using the Sleeper system that we have developed originally. We designed a two-stage culture system in which the stage between for growth and for enzyme production can be separated through a heat exchanger and the culture conditions for each stage can be controlled independently, combined with the Sleeper system. Fermentation performance of the lysogen that harbors the genes for cysteine biosynthesis in the two-stage culture system was compared with the single-stage culture system. In the two-stage culture system we have designed the serine acetyltransferase(SAT) activity reached to 2.5U/mg-protein while lU/mg-protein in case of the single-stage culture.

We present an electron-identification method using the time-projection chamber and the lead-glass calorimeter in the TOPAZ detector system. Using this method we have achieved good electron identification against hadron backgrounds over a wide momentum range in the hadronic events produced by both single-photon exchange and two-photon processes. Pion-rejection factors and electron efficiencies were 163 and 68.4\% for high-$P_T$ electrons and 137 and 42.7\% for low-$P_T$ electrons in the single-photon-exchange process, and 8600 and 36.0\% for the two-photon process, respectively., 32 pages, latex format (article), 24 figures, submitted for publication.

We describe a case in which a left ventricular-coronary sinus communication through a dissecting ventricular septal aneurysm developed after a redo mitral valve replacement. The outlet orifice of the communication was located in the side wall of the ostium of the coronary sinus. Both the communication and the aneurysm were successfully dealt with by performing a right atriotomy and by opening the aneurysm from its outlet orifice.

To search for a new physics and to pin down its interaction model, we start the Belle II experiment in 2015 and will continue the data taking for more than ten years. In the Belle II experiment, sub-detector hit signals are digitized by front-end electronics cards located inside or nearby the detector and the digitized signals are transmitted to receiver cards, which are located ∼ 10 m away from the detector, over optical fiber links. Because of this design, radiation effects to the front-end electronics components will be a severe issue, where the annual neutron flux and γ-ray dose to them are expected to be ∼ 1011/cm2 and ∼ 80 Gy, respectively, in the worst case. We have been carrying out radiation-effect studies by those particles to the front-end electronics components using experimental exposure facilities. In the series of previous studies, we found optical transceivers that we studied were very sensitive to γ rays and had only four-year-equivalent radiation hardness in the Belle II operation. To solve this problem, we search for an optical transceiver with sufficient radiation hardness for Belle II by bombarding several commercial optical transceivers with γ rays. We isolate the AFBR-57D7APZ transceiver (AVAGO) as the one with the highest radiation hardness among the tested products. We quantify the AFBR-57D7APZ transceiver operates until receiving > 900 Gy total γ-ray dose, which is a sufficient radiation hardness for the Belle II use.

Random mutagenesis of the luciferase cDNA from "Genji" firefly, Luciola cruciata, was induced by hydroxylamine in an attempt to isolate thermostable mutants. Three mutants were isolated, and the cDNAs encoding these proteins were sequenced. All mutant cDNAs carried the same C to T transition mutation that conferred an amino acid substitution of Thr by Ile at position 217. The wild-type luciferase and the thermostable variant (Thr217Ile) were purified to homogeneity, and their enzymatic properties were determined. Thr217Ile was superior to wild-type in thermal and pH stability. Furthermore, the specific activity of the Thr217Ile mutant was increased to 130% of that of the wild-type. In order to examine the effect of amino acid residue substitution at position 217 on the thermostability of luciferase, we replaced the Thr residue at position 217 with all of the rest of the possible amino acid residues by site-directed mutagenesis. The thermostability of these substitution mutants seemed to correlate with the hydrophobicity of the substituted amino acid residue.

The sucrose phosphorylase gene was cloned from Leuconostoc mesenteroides ATCC 12291 and was overexpressed in Escherichia coli using a ‘Sleeper’ bacteriophage vector. A recombinant phage slp- spl -1, which had four copies of the sucrose phosphorylase gene, was lysogenized into E. coli 1100. After heat induction, this lysogen produced 55.7 units per ml culture of sucrose phosphorylase, i.e. 80-times higher than that in L. mesenteroides . As the amount of this recombinant enzyme was over 30% of the soluble protein in the cell, E. coli 1100 (slp- spl -1) succeeded in overcoming problems such as, inhibited enzymes, in the case of L. mesenteroides . Thus it is possible to achieve an industrial-scale production of sucrose phosphorylase. The complete nucleotide sequence showed that it coded for a 490-amino acid protein of M T 55,749. Homology between the deduced amino acid sequences for the L. mesenteroides sucrose phosphorylase gene and Streptococcus mutans gtfA , sucrose phosphorylase gene, was 68%.

The genomic DNA for the alkaline protease (Alp) of the fungus Aspergillus oryzae was isolated using synthetic oligonucleotides as hydridization probes, and the complete nucleotide sequence was identified. The Alp gene is 1374 nucleotides long and contains three introns, one of which is in the pro region and two in the mature coding region. Sequences related to the TATA box (TATAAAT) and the CAAT box (CCAAAT) were found in the 5'-noncoding region. Primer extension analysis showed that three transcriptional start points are present.

The neutral protease II (NpII) from Aspergillus oryzae is a zinc-containing metalloprotease with some unique properties. To elucidate its structure, we isolated a full-length cDNA clone for NpII. Sequence analysis reveals that NpII has a prepro region consisting of 175 amino acids preceding the mature region, which consists of 177 amino acids. As compared with other microbial metalloproteases, NpII is found to be unique in that it shares only a limited homology with them around two zinc ligand His residues and that the positions of the other zinc ligand (Glu) and the active site (His) cannot be established by homology. When a plasmid designed to express the prepro NpII cDNA was introduced into Saccharomyces cerevisiae and the transformant was cultured in YPD medium (2% glucose, 2% polypeptone, 1% yeast extract), it secreted a proNpII. However, in a culture of the same medium containing 0.2 mM ZnCl2, it secreted a mature NpII with a specific activity and N-terminus identical to those of native NpII. This observation suggests that either an autoproteolytic activity or a yeast protease effected the processing.

The gene coding for N-acyl-D-mannosamine dehydrogenase (NAM-DH) from Flavobacterium sp. strain 141-8 was cloned and expressed under the control of a lac promoter in Escherichia coli JM109. The DNA sequence of the gene was determined, and an open reading frame encoding a polypeptide composed of 272 amino acid residues (Mr, 27,473) was identified. The E. coli transformants which showed over 200-fold higher NAM-DH activity than did the Flavobacterium strain produced the enzyme as a protein fused with beta-galactosidase. Despite being a fusion, NAM-DH produced by E. coli transformants appeared unchanged in pH optimum, Km, and substrate specificity from Flavobacterium sp. strain 141-8. This newly recombinant enzyme may be applicable to the quantitative determination of sialic acid in serum.

Summary A cDNA clone for mink growth hormone (GH) was isolated from a mink pituitary cDNA library, employing a part of rat growth hormone cDNA sequence as a probe. According to the nucleotide sequence, mature mink GH consists of 190 amino acids with a calculated molecular weight of 21,720. The amino acid sequence homology between the mature region of mink GH and those of pig GH, rat GH, bovine GH and human GH was 98.4%, 93.7%, 89.0% and 66.7%, respectively.

The gene coding for creatinase (creatine amidinohydrolase, EC 3.5.3.3) was isolated from Flavobacterium sp. U-188. The primary structure of creatinase deduced from the nucleotide sequence showed a protein (molecular weight, 42, 651) composed of 378 amino acids. The creatinase gene was over-expressed in Escherichia coli under the control of the lac promoter and the amount of this enzyme was over 20 % of the soluble protein in the cell.

To produce Aspergillus oryzae alkaline protease (Alp) in an osmophilic yeast Zygosaccharomyces rouxii, we constructed an expression plasmid consisting of the Z. rouxii glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter, the prepro-Alp cDNA of A. oryzae, the whole sequence of Z. rouxii plasmid pSR1, and the G418 resistant gene. The resulting plasmid, when introduced into Z. rouxii cells, directed the secretion of a large amount (about 300 mg/l) of Alp into the culture medium. The N-terminus and specific activity of the enzyme were identical to those of A. oryzae Alp.

In every wafer processing step wafer stress management is extremely important for advanced device manufacturing. Thermally induced stress on device wafers has a large impact on lithography and affects device yield. Thermally induced stress during rapid thermal annealing (RTA) steps in high density 512MB DRAM device fabrication was investigated using a lamp-based (cold wall) RTA system and compared to results using a furnace-based (hot wall) single wafer RTA system. Compared to the lamp-based (cold wall) system, RTA in a furnace-based (hot wall) system was found to be very effective in suppressing thermally induced stress and increasing device yield due to superior pattern transfer characteristics in lithography.

The luciferase cDNA from the 'Genji' firefly, Luciola cruciata, was mutated with hydroxylamine to isolate mutant luciferases. Some of the isolated mutant enzymes produced different colors of light, ranging from green to red. Five such mutants, producing green (lambda max = 558 nm), yellow-orange (lambda max = 595 nm), orange (lambda max = 607 nm) and red light (lambda max = 609 and 612 nm), were analyzed. The mutations were found to be single amino acid changes, from Val239 to Ile, Pro452 to Ser, Ser286 to Asn, Gly326 to Ser and His433 to Tyr respectively.

We cloned and analyzed the nucleotide sequence of a cDNA that encodes polyphenol oxidase (laccase) from the white-rot basidiomycete Schizophyllum commune. The nucleotide sequence of the full-length cDNA contained a 1554-base open reading frame that encoded a polypeptide of 518 amino acid residues, including a putative signal peptide of 16 residues. It contained four highly similar regions that are conserved in the deduced amino acid sequences of other laccases, including the region thought to be involved in copper binding. Aspergillus sojae strain 1860 (which has low protease levels) was transformed with the plasmid lacAL/pTPT, which contained the laccase gene under the control of the tannase promoter from Aspergillus oryzae. Laccase was secreted into the medium when transformants A1 and A2 were cultured in tannic acid-containing medium.

A phosphotransacetylase gene (pta) has been cloned from a genomic DNA library of Escherichia coli 1100, a derivative of strain K-12. The phosphotransacetylase activities of pta+ plasmid-containing strains were amplified about 150-fold under control of the lac promoter. The molecular weight of the phosphotransacetylase was estimated to be about 81,000 by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The pta gene was found to be downstream of ackA by a combination of restriction analysis and plasmid subcloning. It is located about 13 kb upstream of the purF-folC-hisT region of the chromosome.

The phosphotransacetylase gene ( pta ) from Escherichia coli strain K-12 1100 was identified in a cloned fragment of chromosomal DNA (Yamamoto-Otake, H., Matsuyama, A. and Nakano, A. (1990) Appl. Microbiol. Biotechnol. 33, 680–682). Overexpression in E. coli confirmed the presence of the pta gene within the cloned fragment. DNA sequence analysis of the cloned pta gene indicates that the predicted phosphotransacetylase polypeptide chain is 713 amino acids in length. The carboxyterminal region of the E. coli phosphotransacetylase shows 42.6% sequence identity with the corresponding enzyme from Methanosarcina thermophila (142 out of 333 residues in corresponding positions are identical). Several short regions of high sequence identity may be structurally or functionally important for enzymic activity.

The neutral proteinase II from Aspergillus oryzae (NpII) is a zinc proteinase with three intramolecular disulfide bonds. NpII is most unstable after 10 min at about 75 degrees C, but regains stability beyond this temperature and is relatively stable at 100 degrees C. We analyzed the thermal stability of wild-type NpII and apo NpII. The results suggested that NpII unfolds reversibly upon incubation up to 100 degrees C, and that the irreversible inactivation observed is mainly due to autoproteolysis. To further understand the stability, a mutant NpII (Cys78-->Ala) lacking one of the disulfide bonds, was produced in a heterologous yeast expression system. The mutant NpII showed a similar stability profile, but the most unstable temperature and the most catalytically active temperature decreased to the same extent (around 10 degrees C), confirming that autoproteolysis is the main cause of the irreversible inactivation. Several lines of evidence presented in this study demonstrated that the thermal stability of o++NpII is attributed to reversible thermal unfolding and autoproteolysis.

The gene for cyclomaltodextrinase (CDase; EC 3.2.1.54) from Bacillus sphaericus E-244 was cloned in the recombinant plasmid pCD629. Sequencing a portion of pCD629 revealed a unique open reading frame of 1,773 nucleotides coding for a 591-amino-acid polypeptide. The deduced polypeptide sequence showed about 50% homology with that of a neopullulanase, and was slightly homologous to those of the cyclodextrin glucanotransferases and the alpha-amylases. The optimum pH, specific activity and Km value for beta-cyclodextrin of the CDase that has been produced in Escherichia coli cells were 8.0, 16.4 units/mg protein, and 0.41 mM, respectively. These values were almost identical to those from B. sphaericus E-244.

We have cloned a cDNA encoding Luciola lateralis (a common firefly in Japan) luciferase from a cDNA library of lantern poly(A) + RNA, using a cDNA of L. cruciata (another common firefly in Japan) luciferase as a probe. The primary structure of L. lateralis luciferase deduced from the nucleotide sequence was shown to consist of 548 amino acids with a molecular weight of 60 132. Sequence comparison indicates that L. lateralis luciferase has significant sequence identity (94%) to L. cruciata luciferase, and that it has less sequence similarity (67%) to Photinus pyralis (a North American firefly) luciferase. The isolated cDNA clone, when introduced into Escherichia coli , directed the synthesis of enzymatically active luciferase under the control of the lacZ promoter.

Luciferases of Luciola cruciata and Luciola lateralis, LcL and LlL, were purified to homogeneity by ammonium sulfate precipitation, gel-filtration column chromatography, and hydroxyapatite HPLC. The molecular masses of the enzymes determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were both 62 kDa, almost identical to that of Photinus pyralis (PpL). LcL was found to be similar to PpL in thermal stability, pH stability, and the wavelength of maximum light intensity. LlL was superior to LcL and PpL in thermal and pH stability, and the reaction catalyzed by LlL emits green light with a peak intensity at 552 nm, which is 10 nm shorter in wavelength than those of PpL and LcL.

The gene coding for a thermostable sarcosine oxidase (EC 1.5.3.1) was isolated from Bacillus sp. NS-129. The primary structure of sarcosine oxidase deduced from the nucleotide sequence was a protein composed of 387 amino acids with molecular weight 42,955. When the sarcosine oxidase was overproduced to about 35% of soluble protein in E. coli under the control of a lac promoter, the sarcosine oxidase activity of the crude extract was increased 3-fold by the addition of FAD. This indicates that most of the enzyme is accumulated in an active form, a flavinless aporotein, in the cell.

The gene coding for L-fucose dehydrogenase (FDH) which is specific to NADP+ as a hydrogen acceptor, was cloned, sequenced, and expressed in Escherichia coli from Pseudomonas sp. No. 1143. FDH was shown to be composed of 329 amino acid residues.

(1992). Enhancement of the Thermostability of the Alkaline Protease from Aspergillus oryzae by Introduction of a Disulfide Bond. Bioscience, Biotechnology, and Biochemistry: Vol. 56, No. 2, pp. 326-327.

We have cloned the cDNA for luciferase from lantern poly(A)+ RNA of a Japanese firefly, Luciola cruciata (Genji botaru in Japanese). This cDNA directed the synthesis of enzymatically active luciferase under the control of the lac promoter in Escherichia coli. The amino acid sequence predicted from the cDNA sequence shows that Genji firefly luciferase consists of 548 amino acids and has a molecular weight of 60,024. Considerable sequence homology was found upon the comparison of the Genji and North American firefly luciferases.

Isolated rat heart preparations were used to determine the effect of cardioplegia on myocardial metabolism during profound hypothermic (15 degrees C) ischemia. The hearts were grouped according to the components of cardioplegia and the mode of administration. The six groups were normokalemic (GI), calcium-containing hyperkalemic (GII), calcium-free hyperkalemic (GIII) and single dose (A), multidose (B). Following 120 min of ischemia, tissue ATP decreased from 25.4 +/- 2.2 to 10.3 +/- 2.7, 3.9 +/- 2.4, 4.1 +/- 1.2, 15.5 +/- 3.2, 14.5 +/- 2.4 and 20.0 +/- 2.7 (I-A vs II-A p less than 0.005, I-A vs III-A p less than 0.005, I-B vs III-B p less than 0.05, II-B vs III-B p less than 0.005), and tissue lactate increased from 9.6 +/- 1.5 to 163.4 +/- 12.0, 174.1 +/- 13.5, 166.8 +/- 21.3, 99.1 +/- 8.3, 102.6 +/- 12.2 and 83.5 +/- 9.3 (I-B vs III-B p less than 0.02, II-B vs III-B p less than 0.02) mumol/dry wt g, in GI-A, GII-A, GIII-A, GI-B and GII-B, respectively. The results of this study suggests that (1) potassium cardioplegia in a single dose does not prevent degradation of high energy phosphate (HEP) in the hypothermic arrested heart, (2) though multidose cardioplegia is effective in preserving HEP during ischemia, the extent of its effects varies with the composition, and (3) the omission of calcium is beneficial in GIK cardioplegia in terms of preserving HEP at the end of ischemia.

A retrospective study of 21 adult patients with tetralogy of Fallot (TOF) was undertaken to determine the predisposing risk factors of renal dysfunction following total correction of the disease. Five of the 21 patients exhibited moderate-to-severe postoperative azotemia and an additional five exhibited mild azotemia. Significant risk factors for postoperative renal dysfunction found in routine preoperative examinations were as follows: arterial oxygen saturation of less than 90%, cardiothoracic ratio (CTR) of greater than 50%, mean electrical axis of the QRS complexes of greater than +120 degrees, S wave in Lead V6 of greater than 7 mm, R/S voltage ratio in Lead V6 of less than 2, and negative T wave in Lead V6. In addition, preoperative cardiac catheterization data showed that the patients exhibiting postoperative azotemia had more severe pulmonary stenosis, a smaller pulmonary-to-systemic flow ratio (Qp/Qs), and a larger left ventricular cavity than nonazotemic patients. The incidence of postoperative low cardiac output state (LOS) was significantly higher in the azotemic patients. These findings suggest that a combination of the severe form of TOF and a large left ventricle increase susceptibility to LOS and postoperative renal dysfunction. The cause and the clinical significance of the large left ventricular cavity in adults with TOF are discussed.

The respiratory-deficient mutant H7, obtained by Beljanski from Monod's strain of Escherichia coil, differs in several respects from the parent strain. Comparison of the DNAs from the two cultures has revealed complete homology. Physiologically, the mutant resembles the homofermentative lactobacilli; it does not contain catalase or cytochromes, grows better in the absence than in the presence of air, produces lactic acid from glucose even under aerobic conditions, and requires amino acids for growth. These properties are discussed in relation to the respiratory deficiency, as is their bearing on the evolutionary status of the lactobacilli.

In 12 dogs being supported by cardiopulmonary bypass, the relationship among myocardial oxygen consumption and four energy-consuming factors (basal metabolism, heart rate, tension development, and external work) was studied. Tension (internal work) in the left ventricular wall was evaluated by myocardial tissue pressure with a Mikro-Tip pressure transducer. In an empty beating heart with constant perfusion pressure, both systolic tissue pressure and developed tissue pressure represented the same characteristics as developed tension measured by other methods. As the heart rate was increased, the systolic tissue pressure and developed tissue pressure continued to increase stepwise (Bowditch effect) up to some stimulation rate, at which, however, a decrease began despite a further increase in heart rate (Woodworth effect). Significant regression was established between myocardial oxygen consumption and heart rate, tension (developed tissue pressure x heart rate), and external work (minute work): myocardial oxygen consumption = (9.05 x 10(-3) heart rate) + (1.95 x 10(-4) developed tissue pressure) x heart rate + (1.63 x 10(-3) minute work) + 1.42 (r = 0.7999), where activation energy = 9.05 x 10(-3) ml/100 gm per beat, tension-related energy = 1.95 x 10(-4) ml/100 gm per unit of internal work, energy for work = 1.63 ml/100 gm per unit of external work, and basal metabolism = 1.42 ml/min/100 gm. We concluded that myocardial tissue pressure is a good substitute for tension and that multiple regression with heart rate, tension, and external work (as by modified Fenn's equation) seems indispensable to predict myocardial oxygen tension in the whole heart.

A plasmid (pMR226) containing the cDNA coding for the complete amino acid sequence of mouse renin was constructed. The cDNA was reconstructed so as to code for either fused mature renin or preprorenin, and inserted into a plasmid designed to express a protein under the control of the Escherichia coli tryptophan promoter. When introduced into E. coli, the resultant plasmids (pMR304, pMR324) directed the synthesis of proteins that cross-reacted with an antiserum raised against purified mouse renin. The renin precursor produced was readily activated on trypsin treatment and the activity was inhibited by pepstatin and antirenin-antibody.