Lead-free MCP to improve coincidence time resolution and reduce MCP direct interactions.

Achieving direct imaging of the annihilation position of a positron on an event-by-event basis using an ultrafast detector would have a great impact on the field of nuclear medicine. Cherenkov emission is the most attractive physical phenomenon for realizing such an ultrafast timing performance. Moreover, a microchannel-plate photomultiplier tube (MCP-PMT) is one of the most promising photodetectors for fully exploiting the fast timing properties of Cherenkov emission owing to its excellent single photon time resolution of 25 ps full width at half maximum (FWHM). However, as the MCP structure generally contains a lead compound, the gamma rays frequently and directly interact with the MCP, resulting in the degradation of its timing performance and generation of undesirable side peaks in its coincidence timing histogram. To overcome this problem, we have developed a new MCP-PMT based on an MCP consisting of borosilicate glass, thus drastically reducing the probability of the photoelectric effect occurring in the MCP. To evaluate its insensitivity to gamma rays and its timing performance, a coincidence experiment was performed and showed that the probability of direct interactions was reduced by a factor of 3.4. Moreover, a coincidence time resolution of 35.4 ± 0.4 ps FWHM, which is equivalent to a position resolution of 5.31 mm, was obtained without any pulse height/area cut, improving to 28.7 ± 3.0 ps when selecting on the highest amplitude events by careful optimization of the voltage divider circuit of the new MCP-PMT. The timing performance of this new MCP-PMT presents an important step toward making direct imaging possible.

Precise analysis of the timing performance of Cherenkov-radiator-integrated MCP-PMTs: analytical deconvolution of MCP direct interactions.

In order to achieve the ultimate goal of reducing coincidence time resolution (CTR) to 10 ps, thus enabling reconstruction-less positron emission tomography, a Cherenkov-radiator-integrated microchannel plate photomultiplier tube (CRI) reaching CTR of sub-50 ps full width at half maximum (FWHM) has been developed. However, a histogram of time differences between a pair of the CRIs shows undesirable side peaks, which are caused by gamma rays directly interacting with the micro channel plates (MCPs). Such direct interaction events are detrimental to the timing performance of the CRI. In this paper, we demonstrate an analytical method of deconvolving MCP direct interaction events from the timing histogram. Considering the information of the main and the two side peaks, the timing uncertainty caused by the MCP direct interaction events is deconvolved and the CTR of the CRI is analytically investigated. Consequently, the CTR is improved from 41.7 to 40.5 ps FWHM by the deconvolution. It means that a mixture of the Cherenkov radiator events and the MCP direct interaction events contribute to the CTR by a factor of 10 ps. The timing performance of the MCP direct interaction events are also evaluated. The CTR between the two MCPs is found to be 66.2 ps FWHM. This indicates that a photocathode-free radiation detector with high timing performance is possible. Elimination of the photocathode from the detector would make detector construction easier and more robust.

Coincidence time resolution of 30 ps FWHM using a pair of Cherenkov-radiator-integrated MCP-PMTs.

Radiation detectors dedicated to time-of-flight positron emission tomography (PET) have been developed, and coincidence time resolution (CTR) of sub-100 ps full width at half maximum (FWHM) has been achieved by carefully optimizing scintillators and photodetectors. Achieving a CTR of 30 ps FWHM by using a pair of annihilation γ-rays would allow us to directly localize the annihilation point within an accuracy of 4.5 mm. Such direct localization can potentially eliminate the requirement of image reconstruction processes in clinical PET systems, which would have a huge impact on clinical protocols and molecular imaging. To obtain such a high CTR, researchers have investigated the use of prompt emissions such as Cherenkov radiation and hot-intra band luminescence. Although it is still challenging to achieve a CTR of 30 ps FWHM even with a Cherenkov-based detector, the experimentally measured CTR is approaching the goal. In this work, we developed a Cherenkov-radiator-integrated micro-channel plate photomultiplier tube (CRI-MCP-PMT), where there are no optical boundaries between the radiator and photocathode, and its timing performance was investigated. By removing the optical boundaries, reflections are eliminated and transmission to the photocathode is improved, resulting in high timing capability. As a result, a CTR of 30.1 ± 2.4 ps FWHM, which is equivalent to a position resolution of 4.5 ± 0.3 mm along a line of response (LOR), was obtained by using a pair of CRI-MCP-PMTs.

Mutations that are synthetically lethal with a gas1Delta allele cause defects in the cell wall of Saccharomyces cerevisiae.

The GAS1-related genes of fungi encode GPI-anchored proteins with beta-1,3-glucanosyltransferase activity. Loss of this activity results in defects in the assembly of the cell wall. We isolated mutants that show a synthetic defect when combined with a gas1Delta allele in Saccharomyces cerevisiae, and identified nine wild-type genes that rescue this defect. The indispensability of BIG1 and KRE6 for the viability of gas1Delta cells confirmed the important role of beta-1,6-glucan in cells that are defective in the processing of beta-1,3-glucan. The identification of the Wsc1p hypo-osmotic stress sensor and components of the PKC signal transduction pathway in our screen also confirmed that the cell wall integrity response attenuates the otherwise lethal gas1Delta defect. Unexpectedly, we found that the KEX2 gene is also required for the viability of the gas1Delta mutant. Kex2p is a Golgi/endosome-membrane-anchored protease that processes secretory preproteins. A cell wall defect was also found in the kex2Delta mutant, which was suppressible by multiple copies of the MKC7 or YAP3 gene, both of which encode other GPI-anchored proteases. Therefore, normal cell wall assembly requires proteolytic processing of secretory preproteins. Furthermore, the genes CSG2 and IPT1 were found to be required for normal growth of gas1Delta cells in the presence of 1 M sorbitol. This finding suggests that complex sphingolipids play a role in the hyper-osmotic response.

Identification of genes required for growth under ethanol stress using transposon mutagenesis in Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae exhibits high ethanol tolerance compared with other microorganisms. The mechanism of ethanol tolerance in yeast is thought to be regulated by many genes. To identify some of these genes, we screened for ethanol-sensitive S. cerevisiae strains among a collection of mutants obtained using transposon mutagenesis. Five ethanol-sensitive (ets) mutants were isolated from approximately 7,000 mutants created by transforming yeast cells with a transposon (mTn-lacZ/LEU2)-mutagenized genomic library. Although these mutants grew normally in a rich medium, they could not grow in the same medium containing 6% ethanol. Sequence analysis of the ets mutants revealed that the transposon was inserted in the coding regions of BEM2, PAT1, ROM2, VPS34 and ADA2. We constructed deletion mutants for these genes by a PCR-directed disruption method and confirmed that the disruptants, like the ets mutants, were ethanol sensitive. Thus, these five genes are indeed required for growth under ethanol stress. These mutants were also more sensitive than normal cells to Calcofluor white, a drug that affects cell wall architecture, and Zymolyase, a yeast lytic enzyme containing mainly beta-1,3- glucanase, indicating that the integrity of the cell wall plays an important role in ethanol tolerance in S. cerevisiae.

Extracellular soluble polysaccharide (ESP) from Aspergillus kawachii improves the stability of extracellular beta-gluocosidases (EX-1 and EX-2) and is involved in their localization.

Aspergillus kawachii produces two extracellular beta-glucosidases (EX-1 and EX-2) and one cell-wall-bound beta-glucosidase (CB-1), all of which are derived from the same bglA gene. Extracellular beta-glucosidases (EX-1 and EX-2) are stable in the crude solution form, but become unstable in the purified form under moderate conditions (pH 5.0 and 37 degrees C). Purified extracellular beta-glucosidases can bind to a mycelial cell wall fraction, even though these enzymes are released into the medium under solid culture conditions. A. kawachii produces an extracellular soluble the beta-glucosidases over the pH range of 3.0-7.0 and at temperatures below 50 degrees C. ESP directly interacted with the purified extracellular beta-glucosidases but did not affect the K(m) values of these enzymes. Moreover, ESP inhibited the adsorption of purified extracellular beta-glucosidases to the cell wall fraction and extracted them from it. These results that ESP plays important roles in the stability and localization of extracellular beta-glucosidases. ESP from A. kawachii directly binds to the enzymes and releases them to the medium from the cell wall layer and then stabilizes them.

Tolerance mechanism of the ethanol-tolerant mutant of sake yeast.

Several ethanol-tolerant mutants have been bred from industrial sake yeasts, but the mechanism of ethanol tolerance in these mutants has not been elucidated. After the determination of the entire genome sequence of Saccharomyces cerevisiae, various methods to monitor the whole-gene expression of the yeast have been developed. In this study, we used a commercially available nylon membrane on which virtually every gene of S. cerevisiae was spotted to compare expression profiles between the ethanol-tolerant mutant and its parent sake yeast to investigate the mechanism of ethanol tolerance in this mutant. As a result, we found that several genes were highly expressed only in the ethanol-tolerant mutant but not in the parent strain. These genes were known to be induced in cells that were exposed to various stresses, such as ethanol, heat, and high osmolarity, or at the stationary-phase but not at the log-phase. In the ethanol-tolerant mutant, the expression level of these stress-responsive genes was further increased after exposure to ethanol. We also found that substances such as catalase, glycerol and trehalose that may have protective roles under stressful conditions were accumulated in high amounts in the ethanol-tolerant mutant. The ethanol-tolerant mutant also exhibited resistance to other stresses including heat, high osmolarity and oxidative stress in addition to ethanol tolerance. These results indicate that the mutant exhibits multiple stress tolerance because of elevated expression of stress-responsive genes, resulting in accumulation of stress protective substances.

Molecular cloning and application of a gene complementing pantothenic acid auxotrophy of sake yeast Kyokai no. 7.

Kyokai no. 7 is the most widely used yeast in sake brewing. This yeast is a pantothenic acid auxotroph at 35 degrees C, and this phenotype has been used to distinguish Kyokai no. 7 from other sake yeasts. We cloned a DNA fragment complementing the pantothenic acid auxotrophy from a genomic library of a Saccharomyces cerevisiae laboratory strain. DNA sequence analysis revealed that the DNA fragment encodes ECM31, the deletion of which had previously been identified as a calcofluor white-sensitive mutation. The ECM31 product is similar to the Escherichia coli ketopantoate hydroxymethyltransferase. Disruption of ECM31 in a laboratory S. cerevisiae strain resulted in pantothenic acid auxotrophy, indicating that ECM31 is also involved in pantothenic acid synthesis in yeast. A hybrid of a Kyokai no. 7 haploid and the ecm31 disruptant required pantothenic acid at 35 degrees C for its growth, suggesting that Kyokai no. 7 possesses a temperature-sensitive allele of ECM31. Thus, the ECM31 gene can be used as a selective marker in the transformation of Kyokai no. 7.

The bglA gene of Aspergillus kawachii encodes both extracellular and cell wall-bound beta-glucosidases.

We cloned the genomic DNA and cDNA of bglA, which encodes beta-glucosidase in Aspergillus kawachii, based on a partial amino acid sequence of purified cell wall-bound beta-glucosidase CB-1. The nucleotide sequence of the cloned bglA gene revealed a 2,933-bp open reading frame with six introns that encodes an 860-amino-acid protein. Based on the deduced amino acid sequence, we concluded that the bglA gene encodes cell wall-bound beta-glucosidase CB-1. The amino acid sequence exhibited high levels of homology with the amino acid sequences of fungal beta-glucosidases classified in subfamily B. We expressed the bglA cDNA in Saccharomyces cerevisiae and detected the recombinant beta-glucosidase in the periplasm fraction of the recombinant yeast. A. kawachii can produce two extracellular beta-glucosidases (EX-1 and EX-2) in addition to the cell wall-bound beta-glucosidase. A. kawachii in which the bglA gene was disrupted produced none of the three beta-glucosidases, as determined by enzyme assays and a Western blot analysis. Thus, we concluded that the bglA gene encodes both extracellular and cell wall-bound beta-glucosidases in A. kawachii.

Structure of the glucan-binding sugar chain of Tip1p, a cell wall protein of Saccharomyces cerevisiae.

Tip1p is one of the major cell wall mannoproteins of Saccharomyces cerevisiae and is presumed to be synthesized as a glycosylphosphatidylinositol (GPI)-anchored form. We purified Tip1p from a glucanase extract of yeast cell walls and analyzed the sugar chain involved in the cell wall linkage. One mol of glucanase-extracted Tip1p contained 7.5 mol of glucose derived from glucan and 1 mol of ethanolamine, a component of the GPI anchor. One mol of the C-terminal peptide of Tip1p digested with Achromobacter protease I also contained 7.9 mol of glucose and 1 mol of ethanolamine. On the other hand, Tip1p contained no glucosamine, which is a component of the GPI anchor. The glucan-binding sugar chain of Tip1p was released by hydrazinolysis and isolated. This sugar chain contained ethanolamine with a free amino group and a glucose reducing end, but no mannose reducing end. Phosphodiesterase treatment eliminated the free amino group from this sugar chain, suggesting that a phosphodiester bond exists between the ethanolamine and the glucan remnant. These results indicate (1) the glucan-binding sugar chain of Tip1p is a GPI derivative, and (2) the GPI anchor is cleaved at the glycosyl moiety, and the resultant mannose reducing end is probably used to link Tip1p to cell wall glucan.

Production and some properties of salt-tolerant beta-xylosidases from a shoyu koji mold, Aspergillus oryzae in solid and liquid cultures.

Beta-xylosidase production from a shoyu (soy sauce) koji mold, Aspergillus oryzae HL15, cultured in solid and liquid media was examined and some properties of the enzymes were studied. Three beta-xylosidases (Xy11, Xy12 and Xy13) were easily extracted with 0.5% NaCl from a solid medium and purified homogeneously on SDS-PAGE by chromatography. On the other hand, in a liquid medium, A. oryzae HL15 produced mainly cell-wall-bound beta-xylosidases which could not be extracted with 0.5% NaCl or any detergent. Cell-wall-bound beta-xylosidases, Xy11-CB and Xy12-CB, were liberated by digestion of mycelia with Yatalase and purified to a homogeneous state on SDS-PAGE by HPLC column chromatography. Four beta-xylosidases (Xy11, Xy12, Xy11-CB and Xy12-CB) exhibited not only high activity at high NaCl concentrations, but also similar properties; on the other hand, Xy13 differed in terms of thermostability and halophilic properties. The salt tolerance of beta-xylosidases in A. oryzae suggests that these enzymes are highly active and involved in releasing xylose in shoyu moromi mash.

Purification and characterization of extracellular and cell wall bound beta-glucosidases from Aspergillus kawachii.

We isolated two extracellular beta-glucosidases (EX-1: 145 kDa, EX-2: 130 kDa) and one cell wall bound beta-glucosidase (CB-1: 120 kDa) from Aspergillus kawachii and characterized their physical and kinetic properties. From the results of N-terminal amino acid sequence, enzymatic parameters and deglycosylation of the three purified enzymes, we strongly suggest that these three enzymes were products of the same gene, modified by different degree of glycosylation. All three purified beta-glucosidases adsorbed to the purified cell wall fraction of this strain. This association could dramatically improve the stability of purified enzymes. These three purified beta-glucosidases were readily inactivated even in moderate conditions but became very stable upon the addition of the purified cell wall fraction. The all purified beta-glucosidases were stable in the range of pH 2.0-9.0 and stable below 30 degrees C with 2 mg/ml of the purified cell wall fraction.

Sed1p is a major cell wall protein of Saccharomyces cerevisiae in the stationary phase and is involved in lytic enzyme resistance.

A 260-kDa structural cell wall protein was purified from sodium dodecyl sulfate-treated cell walls of Saccharomyces cerevisiae by incubation with Rarobacter faecitabidus protease I, which is a yeast-lytic enzyme. Amino acid sequence analysis revealed that this protein is the product of the SED1 gene. SED1 was formerly identified as a multicopy suppressor of erd2, which encodes a protein involved in retrieval of luminal endoplasmic reticulum proteins from the secretory pathway. Sed1p is very rich in threonine and serine and, like other structural cell wall proteins, contains a putative signal sequence for the addition of a glycosylphosphatidylinositol anchor. However, the fact that Sed1p, unlike other cell wall proteins, has six cysteines and seven putative N-glycosylation sites suggests that Sed1p belongs to a new family of cell wall proteins. Epitope-tagged Sed1p was detected in a beta-1,3-glucanase extract of cell walls by immunoblot analysis, suggesting that Sed1p is a glucanase-extractable cell wall protein. The expression of Sed1p mRNa increased in the stationary phase and was accompanied by an increase in the Sed1p content of cell walls. Disruption of SED1 had no effect on exponentially growing cells but made stationary-phase cells sensitive to Zymolyase. These results indicate that Sed1p is a major structural cell wall protein in stationary-phase cells and is required for lytic enzyme resistance.

Identification and analysis of a static culture-specific cell wall protein, Tir1p/Srp1p in Saccharomyces cerevisiae.

A 100-kDa protein was found to be a major cell wall protein in Saccharomyces cerevisiae cells cultured without shaking, but was not present in cells cultured with shaking. The amino acid sequence of this protein was identical to the sequence of Tir1p/Srp1p. TIR1/SRP1 has previously been identified as a gene induced by glucose, cold shock or anaerobiosis and was believed to be a cell membrane protein but not a cell wall protein. However, we found that beta-1,3-glucanase solubilized Tir1p/Srp1p from the cell wall and the purified Tir1p/Srp1p reacted with antiserum to beta-1,6-glucan and contained glucose. These results suggest that Tir1p/Srp1p is a major structural cell wall protein in the static-cultured yeast cells and is bound to the cell wall through beta-1,6-glucan. TIR1/SRP1 mRNA was transcribed only in the static culture and its transcription was regulated by the ROX1 repressor.

Retention of Saccharomyces cerevisiae cell wall proteins through a phosphodiester-linked beta-1,3-/beta-1,6-glucan heteropolymer.

Yeast cell wall proteins, including Cwp1p and alpha-agglutinin, could be released by treating the cell wall with either beta-1,3-or beta-1,6-glucanases, indicating that both polymers are involved in anchoring cell wall proteins. It was shown immunologically that both beta-1,3- and beta-1,6-glucan were linked to yeast cell wall proteins, including Cwp1p and alpha-agglutinin. It was further shown that beta-1,3-glucan was linked to the wall protein through a beta-1,6-glucan moiety. The beta-1,6-glucan moiety could be removed from Cwp1p and other cell wall proteins by cleaving phosphodiester bridges either enzymatically using phosphodiesterases or chemically using ice-cold aqueous hydrofluoric acid. These observations are consistent with the notion that cell wall proteins in Saccharomyces cerevisiae are linked to a beta-1,3-/beta-1,6-glucan heteropolymer through a phosphodiester linkage and that this polymer is responsible for anchoring cell wall proteins. It is proposed that this polymer is identical to the alkali-soluble beta-1,3-/beta-1,6-glucan heteropolymer characterized by Fleet and Manners (1976, 1977).

Molecular cloning of CWP1: a gene encoding a Saccharomyces cerevisiae cell wall protein solubilized with Rarobacter faecitabidus protease I.

A yeast cell wall glycoprotein with a molecular weight of 40,000, named gp40, was solubilized from SDS-extracted cell wall of Saccharomyces cerevisiae by incubation with Rarobacter faecitabidus protease I, which is a yeast-lytic enzyme. Based on its amino acid sequence, we cloned and sequenced the gene encoding the precursor of gp40, named CWP1; cell wall protein gene. The DNA sequence of the CWP1 gene was identical to YKL443, an open reading frame identified in a genome sequencing program for yeast chromosome XI. This gene encoded a serine-rich protein of 239 amino acids with a molecular weight of 24,267. The presence of hydrophobic sequences in the N- and C-termini of the CWP1 protein suggests that it is secreted as a glycosylphosphatidylinositol-anchored protein and is subsequently integrated into the cell wall. Since a gene disruption experiment showed no growth defect, the CWP1 gene is not essential for growth. Mutant CWP1 protein deficient in the C-terminal hydrophobic sequence was secreted into the culture medium, not anchored to the cell wall, thereby indicating that this hydrophobic sequence plays a crucial role in anchoring to the cell wall. Homology between the CWP1 protein and TIP1 family of cold shock proteins suggests that they belong to a new family of cell wall proteins.

Tissue-specific molecular diversity of amidating enzymes (peptidylglycine alpha-hydroxylating monooxygenase and peptidylhydroxyglycine N-C lyase) in Xenopus laevis.

We investigated the molecular diversity of the paired enzymes, peptidylglycine alpha-hydroxylating monooxygenase (PHM) and peptidylhydroxyglycine N-C lyase (PHL), involved in peptide C-terminal amidation. Three kinds of amidating enzyme (AE) cDNAs (AE-I, AE-II and AE-III) have previously been isolated from Xenopus laevis skin. While AE-I cDNA encodes only PHM, AE-III cDNA encodes a protein containing both PHM and PHL sequences and a transmembrane domain. On the other hand, the translated product of AE-II has not been detected yet. Endoproteolytic cleavage of the AE-III protein generates separated forms of PHM and PHL that are purified from X. laevis skin. Expression of AE-III in insect cells using a baculovirus expression vector system indicated that PHM and PHL exist as a membrane-associated, bifunctional enzyme without endoproteolysis in insect cells. Both PHM and PHL activities were detected in all the X. laevis tissues examined. Particularly, the highest levels of both activities were found in skin, brain and heart. We identified basically three types of enzymes in X. laevis; soluble PHM, soluble PHL and a membrane-associated, bifunctional enzyme that has both PHM and PHL domains. While the skin contained soluble types of PHM and PHL, the brain and heart predominantly contained the membrane-associated, bifunctional type. Analysis of mRNA levels by the reverse-transcript polymerase chain reaction method and Western blot analysis using PHM-specific antibody revealed that such molecular diversity of PHM and PHL among the tissues are produced by changing the ratio of AE-I mRNA/AE-III mRNA, and by endoproteolytic processing of the membrane-associated precursor protein.

Molecular structure of Rarobacter faecitabidus protease I. A yeast-lytic serine protease having mannose-binding activity.

Rarobacter faecitabidus protease I (RPI) is a serine protease exhibiting lytic activity toward living yeast cells. RPI is similar to elastase in its substrate specificity and has a lectin-like affinity for mannose. The gene encoding RPI was cloned to elucidate its structure and function. And its nucleotide sequence revealed that it contains an open reading frame encoding a 525-amino acid protein. Homology comparison indicated that pre-pro-RPI consists of three domains: (1) an NH2-terminal prepro domain not found in the mature form of RPI, (2) a protease domain homologous to the trypsin family of serine proteases, and (3) a COOH-terminal domain homologous to the COOH-terminal part of Oerskovia xanthineolytica beta-1,3-glucanase and the NH2-terminal part of the ricin B chain, a lectin isolated from the part of the ricin B chain, a lectin isolated from the castor bean. The RPI gene and its mutant were subsequently expressed in Escherichia coli under its beta-galactosidase promoter to investigate the function of the COOH-terminal domain. The mutant RPI, whose COOH-terminal domain was truncated by site-directed mutagenesis, lost both its mannose-binding and yeast-lytic activity, although the protease activity was not affected. These findings suggest that the COOH-terminal domain actually participates in the mannose-binding activity and is required for yeast-lytic activity.

Characterization of a Xenopus laevis skin peptidylglycine alpha-hydroxylating monooxygenase expressed in insect-cell culture.

The C-terminal amide structure of peptide hormones and neurotransmitters is synthesized via a two-step reaction catalyzed by peptidylglycine alpha-hydroxylating monooxygenase (PHM) and peptidylhydroxyglycine N-C lyase. A Xenopus laevis PHM expressed in insect-cell culture by the baculovirus-expression-vector system was purified to homogeneity and characterized. Using a newly established assay system for PHM, the kinetic features of this enzyme were investigated. As expected, the enzyme required copper ions, L-ascorbate and molecular oxygen for turnover. Salts like KI and KCl, and catalase stabilized the enzyme in the presence of L-ascorbate. The optimum pH value for the enzyme reaction was around six when Mes buffer was used and around seven when phosphate buffer was used under the same assay condition. Below pH 6, acetate, iodide and chloride ions activated the reaction. The kinetic analysis is consistent with a ping-pong mechanism with respect to peptide and L-ascorbate, and the peptide showed substrate inhibition. The substrate specificity of the enzyme at the penultimate position was examined by competitive assay using tripeptides with glycine at the C-termini and the inhibitory potency of these peptides in descending order was methionine > aromatic > non-polar amino acids.