Identification of novel direct transcriptional targets of glucocorticoid receptor.

Transcription of the genes Granzyme A (GZMA), FK506 binding protein 51 (FKBP5), and Down syndrome critical region gene 1 (DSCR1) is upregulated in leukemic cells upon treatment with glucocorticoids (GCs). Several lines of evidence suggest that these genes are implicated in GC-induced apoptosis upstream of the Bcl-2 family of proteins. These genes were upregulated by GC even in the presence of an inhibitor of protein synthesis, cycloheximide, indicating that they are direct target genes of glucocorticoid receptors. DSCR1 is reported to have four isoforms, each of which has a distinct first exon, E1-E4. Among these isoforms, the one with E1 was selectively upregulated by GC. GZMA and FKBP5 have a cluster of putative glucocorticoid response elements (GREs) in introns 1 and 2, respectively, that was identified to be responsible for the response to GC. They were composed of one complete (A/T)G(A/T)(A/T)C(A/T) sequence surrounded by two incomplete (A/T)G(A/T)(A/T)C(A/T) sequences separated by one to four nucleotides. DSCR1, however, did not have a functional GRE upstream or downstream of exon 1. These studies may lead to improved therapeutic uses of GCs in leukemia and lymphoma based upon the expression of these GC target genes.

Modification of autolysis by synthetic peptides derived from the presumptive binding domain of Staphylococcus aureus autolysin.

The autolytic cell wall hydrolase of Staphylococcus aureus, Atl, contains three highly cationic repeats in the central region of the amino acid sequence, and the repeats are presumed to have the role of binding the enzyme to some components on the cell surface. To explain the possible function of the repeats, we synthesized a number of 10- to 30-mer oligopeptides based on the Atl amino acid sequence (Thr432-Lys610) containing repeat 1, and examined their effects on the autolysis of S. aureus cells. When the peptides were added to a cell suspension of S. aureus under low ionic strength conditions, five peptides, A10, A11, A14, A16 and B9, showed immediate increases in optical density (OD) of the cell suspension accompanied by decreases in viable cell counts. After the immediate increases, the ODs for A10 and A14 changed little in the first 2 hr. In contrast, the ODs for A11 and A16 decreased rapidly. When peptide A10 was added to suspensions of heat-killed whole cells, crude cell walls and a crude peptidoglycan preparation, their ODs were increased approximately 2-fold. In contrast, the OD was not increased when the peptide was added to a suspension of pure peptidoglycan from which anionic polymers had been removed. Light microscopic and transmission electron microscopic study showed that A10 and A14 inhibited autolysis and that A11 and A16 induced autolysis earlier than the control. These results suggest strongly that the peptides adsorb to and precipitate on the anionic cell surface polymers such as teichoic acid and lipoteichoic acid via ionic interaction. The effects of peptides on the autolysis may be the results of the modification of S. aureus autolysin activities. These peptides, especially the 10-mer peptide B9 (PGTKLYTVPW) that represents the C-terminal half of A10 and N-terminal half of A11, may be important segments for Atl to bind to the cell surface.

Automated DNA fragment collection by capillary array gel electrophoresis in search of differentially expressed genes.

An automatic DNA fragment collector using capillary array gel electrophoresis has been developed. A sheath flow technique is used for not only detection but also collection of DNA fragments. In a sheath flow cell, the DNA fragments separated by 16 capillaries flow independently into corresponding sampling capillaries. The fraction collector consists of 16 sampling trays and each sampling tray is set beneath each end of the sampling capillaries to collect the flow-through DNA fragments. Certain DNA fragments are automatically sorted by controlling the movement of the sampling trays according to the signals from the system. The collector experimentally separated two mixtures of polymerase chain reaction (PCR) products: one prepared by using eight different sizes (base lengths from 161 to 562) of DNAs; and the other prepared by a differential display (DD) method with cDNA fragments. Collected DNA fragments are amplified by PCR and measured by electrophoresis. DNA fragments with base length differences of one (base lengths 363 and 364) were successfully separated. A separated DNA fragment from the DD sample was also successfully sequenced. In addition, differentially expressed DNA fragments were automatically sorted by comparative analysis, in which two similar cDNA fragment groups, labeled by two different fluorophores, respectively, were analyzed in the same gel-filled capillary. These results show that the automatic DNA fragment collector is useful for gene hunting in research fields such as drug discovery and DNA diagnostics.

Phylogenetic Relationships among Asian species of Petaurista (Rodentia, Sciuridae), Inferred from Mitochondrial Cytochrome b Gene Sequences.

To elucidate the phylogenetic relationships among four species belonging to the genus Petaurista (P. alborufus castaneus, P. alborufus lena, P. leucogenys leucogenys, P. leucogenys nikkonis, P. petaurista melanotus, and P. philippensis grandis), we investigated the partial sequences (1,068 bp) of the mitochondrial cytochrome b gene for these giant flying squirrels. Phylogenetic trees (NJ, MP, and ML trees) constructed from cytochrome b sequences indicated that P. leucogenys was grouped independently with other species, and that P. philippensis was most closely related to P. petaurista with 99-100% bootstrap values. In addition, two subspecies of P. alborufus did not form a single clade: P. alborufus castaneus from China was most distantly related to the other species, whereas P. alborufus lena from Taiwan was closely related to P. petaurista and P. philippensis with 82-90% bootstrap values. This result suggests that it is reasonable to regard P. alborufus lena as a distinct species from P. alborufus castaneus.

Phylogeny and zoogeography of six squirrel species of the genus sciurus (mammalia, rodentia), inferred from cytochrome B gene sequences.

To investigate the phylogenetic relationships between the New World Sciurus and the Old World Sciurus and their biogeographic history, the partial mitochondrial cytochrome b gene sequences (1,040 base pairs) were analyzed on six Sciurus species: S. aberti, S. carolinensis, S. lis, S. niger, S. stramineus, and S. vulgaris. Phylogenetic trees (maximum parsimony, neighbor-joining, and maximum likelihood methods) commonly showed two groups with high bootstrap values (73-100%): one consisting of the New World Sciurus and the other consisting of the Old World Sciurus. Genetic distances among the New World Sciurus species were remarkably larger than that between two Sciurus species of the Old World, suggesting the earlier radiation of the New World Sciurus than the Old World Sciurus.

Potent bacteriolytic activity of ritipenem associated with a characteristic profile of affinities for penicillin-binding proteins of Haemophilus influenzae.

Ritipenem is highly bacteriolytic against Haemophilus influenzae. Bacterial lysis was shown after treatments with ritipenem and cefsulodin at their MICs and after treatments with fropenem and cefdinir at four times their MICs, indicated by decreases in the culture turbidities and by morphological changes of the destroyed cells. These beta-lactams were preferentially bound to penicillin-binding protein (PBP) 1b. Ritipenem, fropenem, and cefsulodin exhibited poor affinities to PBPs 3a and 3b, but cefdinir showed high affinities to these PBPs. Microscopic examinations revealed that selective PBP 3 inhibitors, such as aztreonam and cefotaxime, inhibited lysis induced by ritipenem. These results suggest that the preferential inactivation of PBP 1b could be essential to induce the lysis of H. influenzae cells and that binding to PBPs 3a and 3b may interfere with lysis.

Expression analysis of the autolysin gene (atl) of Staphylococcus aureus.

The bifunctional autolysin gene (atl) of Staphylococcus aureus was transcribed into a 4.1-kb transcript. The transcription initiation site was located at an adenine residue 33-nt upstream from the putative atl start codon. Analysis using a promoter-reporter plasmid showed that promoter activity increased during the exponential growth phase. The Tn551 insertion site of the autolysis-deficient mutant S. aureus RUSAL2 was located in the putative catalytic region of the glucosaminidase domain.

Identification of DNA gyrase inhibitor (GyrI) in Escherichia coli.

DNA gyrase is an essential enzyme in DNA replication in Escherichia coli. It mediates the introduction of negative supercoils near oriC, removal of positive supercoils ahead of the growing DNA fork, and separation of the two daughter duplexes. In the course of purifying DNA gyrase from E. coli KL16, we found an 18-kDa protein that inhibited the supercoiling activity of DNA gyrase, and we coined it DNA gyrase inhibitory protein (GyrI). Its NH2-terminal amino acid sequence of 16 residues was determined to be identical to that of a putative gene product (a polypeptide of 157 amino acids) encoded by yeeB (EMBL accession no. U00009) and sbmC (Baquero, M. R., Bouzon, M., Varea, J., and Moreno, F. (1995) Mol. Microbiol. 18, 301-311) of E. coli. Assuming the identity of the gene (gyrI) encoding GyrI with the previously reported genes yeeB and sbmC, we cloned the gene after amplification by polymerase chain reaction and purified the 18-kDa protein from an E. coli strain overexpressing it. The purified 18-kDa protein was confirmed to inhibit the supercoiling activity of DNA gyrase in vitro. In vivo, both overexpression and antisense expression of the gyrI gene induced filamentous growth of cells and suppressed cell proliferation. GyrI protein is the first identified chromosomally nucleoid-encoded regulatory factor of DNA gyrase in E. coli.

Localized perforation of the cell wall by a major autolysin: atl gene products and the onset of penicillin-induced lysis of Staphylococcus aureus.

We investigated the cell surface localization of the atl gene products of Staphylococcus aureus exposed to a lytic concentration (4 MIC) of penicillin G (PCG) by means of immunoelectron microscopy using anti-62-kDa N-acetylmuramyl-L-alanine amidase or anti-51-kDa endo-beta-N-acetylglucosaminidase immunoglobulin G. Protein A-gold conjugates reacting with antigen-antibody complex localized at sites of defects of the cell wall at the nascent cross wall. Anti-62-kDa N-acetylmuramyl-L-alanine amidase or anti-51-kDa endo-beta-N-acetylglucosaminidase immunoglobulin G inhibited the decreased turbidity caused by PCG-induced lysis and the formation of defects in the wall. The autolysis-defective mutant, S. aureus RUSAL2 (atl::Tn551), exposed to 4 MIC of PCG resisted autolysis and formation of the wall defect. These results suggest that activation or deregulation of the atl gene products at localized sites where formation of new cross wall was disturbed by PCG causes small defects in the cell wall in situ, eventually leading to general autolysis.

Subcellular localization of the major autolysin, ATL and its processed proteins in Staphylococcus aureus.

The Staphylococcus aureus autolysin gene, atl, encodes a unique 138-kDa protein (ATL) with amidase and glucosaminidase domains. ATL has been suggested to undergo proteolytic processing to generate two extracellular peptidoglycan hydrolases, 51-kDa endo-beta-N-acetylglucosaminidase (51-kDa GL) and 62-kDa N-acetylmuramyl-L-alanine amidase (62-kDa AM). To investigate cell-associated bacteriolytic enzymes for atl gene products, proteins were extracted from the cells as follows. The cells were exposed to 3 M LiCl followed by 4% SDS. Thereafter, the cells were disrupted and again extracted with 4% SDS. Whole SDS-stable cell-associated bacteriolytic proteins were extracted without disrupting the cells. Exposure to 3 M LiCl released major 138-, 115-, 85-, 62- and 51-kDa bacteriolytic proteins, and subsequent 4% SDS extraction released major 138- and 115-kDa bacteriolytic proteins. These bacteriolytic proteins were missing in extracts of atl mutant RUSAL2 (S. aureus RN450 atl::Tn551). Immunoblotting studies suggest that these are all atl gene products: the 138-kDa protein is an ATL with a cleaved signal sequence; the 115- and 85-kDa proteins are intermediates; and the 51- and 62-kDa proteins are cell-associated 51-kDa GL and 62-kDa AM, respectively. The trypsin susceptibility of these proteins suggests that they are located outside the cell membrane. Differences in extractability and immunoelectron microscopic studies suggest that atl gene products are associated with cells in two different ways, LiCl extractable and non extractable. We suggest that the 138-kDa ATL undergoes processing through intermediate proteins (115- and 85-kDa proteins) to mature as the active cell cluster-dispersing enzymes 51-kDa GL and 62-kDa AM on the cell surface.

Phylogenetic relationships among Japanese species of the family Sciuridae (Mammalia, Rodentia), inferred from nucleotide sequences of mitochondrial 12S ribosomal RNA genes.

In order to investigate phylogenetic relationships of the family Sciuridae living in Japan, we sequenced partial regions (379 bases) of mitochondrial 12S rRNA genes in six species of Japanese and other Asian squirrels. Phylogenetic trees constructed by sequence data indicated that two genera of flying squirrels (Petaurista and Pteromys) were clustered in a group distinct from non-flying squirrels, suggesting a possible monophyletic relationships of these flying squirrels. The evolutionary distance between the Japanese squirrel (Sciurus lis) from Honshu island and the Eurasian red squirrel (Sciurus vulgaris) from Hokkaido island was comparable to intraspecific distances of the remaining species examined.

An autolysin ring associated with cell separation of Staphylococcus aureus.

atl is a newly discovered autolysin gene in Staphylococcus aureus. The gene product, ATL, is a unique, bifunctional protein that has an amidase domain and a glucosaminidase domain. It undergoes proteolytic processing to generate two extracellular peptidoglycan hydrolases, a 59-kDa endo-beta-N-acetylglucosaminidase and a 62-kDa N-acetylmuramyl-L-alanine amidase. It has been suggested that these enzymes are involved in the separation of daughter cells after cell division. We recently demonstrated that atl gene products are cell associated (unpublished data). The cell surface localization of the atl gene products was investigated by immunoelectron microscopy using anti-62-kDa N-acetylmuramyl-L-alanine amidase or anti-51-kDa endo-beta-N-acetylglucosaminidase immunoglobulin G. Protein A-gold particles reacting with the antigen-antibody complex were found to form a ring structure on the cell surface at the septal region for the next cell division site. Electron microscopic examination of an ultrathin section of the preembedded sample revealed preferential distribution of the gold particles at the presumptive sites for cell separation where the new septa had not been completed. The distribution of the gold particles on the surface of protoplast cells and the association of the gold particles with fibrous materials extending from the cells suggested that some atl gene products were associated with a cellular component extending from the cell membrane, such as lipoteichoic acid. The formation of a ring structure of atl gene products may be required for efficient partitioning of daughter cells after cell division.

Identification of endo-beta-N-acetylglucosaminidase and N-acetylmuramyl-L-alanine amidase as cluster-dispersing enzymes in Staphylococcus aureus.

Two proteins which are capable of dispersing cell clusters of Staphylococcus aureus have been purified from a S. aureus FDA209P culture supernatant. Both of them were found to have bacteriolytic activity. From the elution profile of column chromatography and Western blot (immunoblot) analysis, one of them was identified as a 51-kDa endo-beta-N-acetylglucosaminidase (GL). The other was a 62-kDa protein on the basis of sodium dodecyl sulfate gel electrophoresis. Analysis of the peptidoglycan fragments following treatment with the 62-kDa protein indicated that this protein is an N-acetylmuramyl-L-alanine amidase (AM). In vitro studies of cluster dispersion activities using S. aureus mutant strains Lyt66 or S. aureus Wood46 grown as clusters demonstrated that these two enzymes act synergistically to disperse clusters into single cells. Antiserum against the 51-kDa GL cross-reacted with the 62-kDa AM, and S. aureus FDA209P grown in the presence of anti-51-kDa-GL immunoglobulin G induced giant clusters. Clusters induced by anti-51-kDa GL and by Cibacron blue F3G-A were dispersed by coincubation with the 51-kDa GL and the 62-kDa AM. Western blot analysis demonstrated that the 51-kDa GL and the 62-kDa AM were missing in culture supernatants of S. aureus Lyt66, Wood46, and RUSAL2 (Tn551 autolysin-defective mutant), which grow in clusters. These results strongly suggest that the 51-kDa GL and 62-kDa AM are involved in cell separation of daughter cells after cell division.

Cloning, sequence analysis, and chromosomal assignment of the mouse Apex gene.

APEX nuclease (Apex gene product) is a mammalian multifunctional DNA repair enzyme possibly involved in the repair of apurinic/apyrimidinic (AP) sites and single-strand DNA breaks with 3' termini blocked by nucleotide fragments and also in transcriptional regulation via redox activation of the AP-1 transcription factors. We cloned a 15-kb DNA fragment containing the Apex gene from a mouse leukocyte genomic library and determined a 4-kb stretch of its nucleotide sequence, including the complete sequence of the mouse Apex gene. The gene consists of 5 exons and 4 introns spanning 2.21 kb, and the boundaries between exons and introns follow the GT/AG rule. Two major and one minor transcription initiation sites were assigned to positions +1 and +24 and position +14, respectively, by a combination of ribonuclease protection, primer extension, and 5' RACE analyses. Position +1 is located 312 nucleotides upstream from the ATG initiation codon. The translation initiation and termination sites are located in exon II and exon V, respectively. The sequenced 5' flanking region (1.32 kb) lacks a typical TATA box, but contains a CAAT box and putative binding sites for several transcription factors, such as ATF, NF-IL6, Sp1, and AP2. The 0.8-kb region from position -410 (5' flanking region) to position +386 (intron II) contains a CpG island. The Apex gene locus was mapped to mouse chromosome 14C2-D1 using in situ hybridization.

A Staphylococcus aureus autolysin that has an N-acetylmuramoyl-L-alanine amidase domain and an endo-beta-N-acetylglucosaminidase domain: cloning, sequence analysis, and characterization.

The Tn551 insertion site of the autolysis-deficient Staphylococcus aureus mutant RUSAL2 was cloned and used to identify the autolysis gene atl in the parent strain, RN450. The open reading frame for atl was 3768 bp in length, encoding a deduced protein of 1256 amino acids and molecular size of 137,381 Da. The atl gene product is a bifunctional protein that has an amidase domain and an endo-beta-N-acetylglucosaminidase domain which must undergo proteolytic processing to generate the two extracellular lytic enzymes found in the culture broth of S. aureus.

Physico-chemical and processing quality of porcine M. longissimus dorsi frozen at different temperatures.

Longissimus dorsi muscle from six pigs (24 h post-mortem) was cut into portions of similar size and shape (c. 700 g) and vacuum-packed in polyfilm. The muscle specimens were divided into three samples, one frozen at -20°C, another at -80°C and the third served as the control (not frozen). The meat sample frozen at -80°C was transferred to the -20°C freezer. After one month, both frozen pork samples were thawed at -2°C and drip loss (%) was measured. Hunter colour, metmyoglobin (MetMb) formation (%), water-holding capacity (WHC), TBA value, transmission value (TM) and myofibril fragmentation were also determined. There was no significant difference in drip loss for the two frozen samples. No MetMb formation could be detected and Hunter values were basically the same for all three samples. WHC, TBA value and TM were essentially the same for all three samples. TBA value was quite low for each frozen sample, indicating that lipid oxidation did not occur during freezing. Histological examination of both frozen samples indicated inter- and intracellular ice crystal formation at -20°C, and intracellular ice at -80°C, the extent being less than at -20°C. At -20°C, ice crystals were larger and muscle fibre diameter smaller than for the control or -80°C sample. Myofibril fragmentation in both frozen samples was significantly higher than in the control. Pork sausage was prepared from all three samples by adding 2% NaCl and 100 ppm NaNO(2). Cooking loss and colour forming ratios were essentially the same. The sausage sample made from the -20°C frozen meat was harder than that of the other two samples according to rheological measurement.

Structure, promoter analysis and chromosomal assignment of the human APEX gene.

APEX nuclease is a mammalian DNA repair enzyme having apurinic/apyrimidinic endonuclease, 3'-5'-exonuclease, DNA 3' repair diesterase and DNA 3'-phosphatase activities. This report describes the organization of the gene (APEX gene) for human APEX nuclease. Human APEX gene was cloned using human APEX cDNA and a human leukocyte genomic library in bacteriophage vector EMBL-3. We proved that human APEX gene consists of 5 exons spanning 2.64 kilobases and suggested that the gene exists as a single copy in the haploid genome. The boundaries between exon and intron follow the GT/AG rule. The major transcription initiation site was assigned by primer extension analysis to C at 515 nucleotides upstream from the ATG initiation codon. The translation initiation and termination sites locate in the exon II and V, respectively. The 5' flanking region (0.89 kilobase) sequenced lacks typical TATA and CAAT boxes, but contains TATA- and CAAT-like sequences and putative cis-acting regulatory elements such as binding sites for Sp1, AP2 and ATF. A part of the 5' flanking region belongs to a CpG island, which extends to the intron II. The CpG island is thought to be a transcription regulatory region of APEX gene, a housekeeping gene. The promoter activity of the 5' upstream region was analyzed by introducing the region in HeLa cells in an expression construct containing luciferase gene as a reporter gene, and the region from position 130 bp upstream to position 205 bp downstream of the major transcription initiation site was shown to be enough for high promoter activity. Northern hybridization experiments suggested that the gene is expressed ubiquitously in human cells. The locus of APEX gene was mapped to human chromosome 14q11.2-q12 using the in situ hybridization technique.

Developmental changes in paramesonephric and mesonephric ducts and the external genitalia in swine fetuses during sexual differentiation.

The paramesonephric (Müllerian) duct was first observed in the vicinity of the mesonephric (Wolffian) duct in 30-day-old swine fetuses of both sexes at the level close to the gonad. The paramesonephric duct extended caudally in parallel with the mesonephric duct on day 35 of gestation. By day 40 the paramesonephric duct reached the urogenital sinus. At this stage, the paramesonephric duct began to degenerate in the male, while it continued to develop in the female. This suggests that an anti-Müllerian duct hormone (AMH) is produced before day 40 of gestation. By day 45 of gestation, the mesonephric duct began to decrease in diameter and was accompanied with the involution of the mesonephros in both sexes. By day 60, the male and female mesonephric ducts reduced in their diameter by 70%. Thereafter, the female mesonephric ducts disappeared, while the male ducts developed again. The sex differences was first observed on day 35 in the differentiation of the external genitalia when a small circular urogenital orifice and the anogenital raphe appeared at the sites caudal to the genital tubercle in the male. Such structures were not present in the female. These results suggest that the fetal pig testis is activated to secrete androgen before day 35 of gestation.

Effect of glucose on thioltransferase activity and protein mixed disulfides concentration in GSH-depleting reagents treated rat erythrocytes.

Thioltransferase activity in rat erythrocytes is decreased and the protein mixed disulfide-concentration is increased by the treatment of t-butylhydroperoxide or diamide, significantly. Their levels are restored to their original levels very effectively by the addition of glucose.

Isolation and characterization of a Tn551-autolysis mutant of Staphylococcus aureus.

A Lyt- mutant with reduced autolytic activity was isolated after Tn551 mutagenesis of the methicillin-susceptible Staphylococcus aureus laboratory strain RN450. The Lyt- phenotype could be transferred back into the parent and into a variety of other S. aureus strains by transduction of the transposon marker. Southern analysis has located the Tn551 insert to a 3.2-kb HindIII DNA fragment on the SmaI B fragment of the staphylococcal chromosome. The Lyt- phenotype included reduced rates of cell wall turnover and autolysis induced by detergent or methicillin treatment; however, the rate of methicillin-induced killing was not affected. Peptidoglycans prepared from the parental and mutant cells showed identical muropeptide compositions, as resolved by a high-resolution high-pressure liquid chromatography technique. On the other hand, LiCl extracts of the mutant cells contained reduced amounts of total protein and lower specific cell wall-degrading activity compared with those of extracts of parental cells. The profile of bacteriolytic enzymes as detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed multiple band differences between mutant and parental cells; a major lytic band with properties characteristic of the staphylococcal endo-beta-N-acetylglucosaminidase was completely absent from the Lyt- cells. The Lyt- phenotype transduced into a series of methicillin-resistant strains of both homogeneous and heterogeneous phenotypes caused only a modest decrease in the level of methicillin resistance, as determined by population analysis.