Sleeping beauty transposon-based phenotypic analysis of mice: lack of Arpc3 results in defective trophoblast outgrowth

The Sleeping Beauty (SB) transposon system has generated many transposon-insertional mutant mouse lines, some of which have resulted in embryonic lethality when bred to homozygosity. Here we report one such insertion mapped to the mouse actin-related protein complex subunit 3 gene (Arpc3). Arpc3 is a component of the Arp2/3 complex, which plays a major role in actin nucleation with Y-shaped branching from the mother actin filament in response to migration signaling. Arpc3 transposon-inserted mutants developed only to the blastocyst stage. In vitro blastocyst culture of Arpc3 mutants exhibited severe spreading impairment of trophoblasts. This phenotype was also observed in compound heterozygotes generated using conventional gene-targeted and transposon-inserted alleles. Arpc3-deficient mutants were shown to lack actin-rich structures in the spreading trophoblast. Electron microscopic analysis demonstrated the lack of mesh-like structures at the cell periphery, suggesting a role of Arpc3 in Y-shaped branching formation. These data indicate the importance of Arpc3 in the Arp2/3 complex for trophoblast outgrowth and suggest that Arpc3 may be indispensable for implantation.     

Inhibition of class C beta-lactamases: structure of a reaction intermediate with a cephem sulfone

The crystallographic structure of the Enterobacter cloacae GC1 extended-spectrum class C beta-lactamase, inhibited by a new 7-alkylidenecephalosporin sulfone, has been determined by X-ray diffraction at 100 K to a resolution of 1.6 A. The crystal structure was solved by molecular replacement using the unliganded structure [Crichlow et al. (1999) Biochemistry 38, 10256-10261] and refined to a crystallographic R-factor equal to 0.183 (R(free) 0.208). Cryoquenching of the reaction of the sulfone with the enzyme produced an intermediate that is covalently bound via Ser64. After acylation of the beta-lactam ring, the dihydrothiazine dioxide ring opened with departure of the sulfinate. Nucleophilic attack of a side chain pyridine nitrogen atom on the C6 atom of the resultant imine yielded a bicyclic aromatic system which helps to stabilize the acyl enzyme to hydrolysis. A structural assist to this resonance stabilization is the positioning of the anionic sulfinate group between the probable catalytic base (Tyr150) and the acyl ester bond so as to block the approach of a potentially deacylating water molecule. Comparison of the liganded and unliganded protein structures showed that a major movement (up to 7 A) and refolding of part of the Omega-loop (215-224) accompanies the binding of the inhibitor. This conformational flexibility in the Omega-loop may form the basis of an extended-spectrum activity of class C beta-lactamases against modern cephalosporins.     

Transient in vivo reporter gene assay for ecdysteroid action in the Bombyx mori silk gland

To analyze the molecular mechanisms underlying hormone-regulated gene expression during molt and metamorphosis, we developed a transient reporter gene assay system using the silkworm anterior silk gland. Reporter plasmids were delivered into dissected anterior silk glands by particle bombardment and bombarded glands transplanted into other larvae, to which hormones were then administered. When the green fluorescent protein gene, coupled with the constitutive cytoplasmic actin gene A3 promoter, was introduced into the anterior silk gland, strong green fluorescence was observed a few days later. Bombarded silk glands transplanted into other larvae showed the same morphological changes as intrinsic glands after 20-hydroxyecdysone (20E) alone or 20E plus juvenile hormone (JH) treatment, indicating that the transplanted gland received hormonal signals properly. When a 20E-responsive reporter construct containing four tandemly repeated pal-1 ecdysone response elements upstream from the luciferase gene was delivered into the gland, an approximately 50-fold increase in luciferase activity was detected 30 h after 20E injection. This induction was comparable to that in an ecdysteroid-responsive Bombyx cell line. This in vivo reporter assay system is thus a rapid, effective tool for analyzing gene expression regulated by 20E and probably by JH.     

Inhibition of class A and class C beta-lactamases by penems: crystallographic structures of a novel 1,4-thiazepine intermediate

A new beta-lactamase inhibitor, a methylidene penem having a 5,6-dihydro-8H-imidazo[2,1-c][1,4]oxazine heterocyclic substituent at the C6 position with a Z configuration, irreversibly inhibits both class A and class C serine beta-lactamases with IC(50) values of 0.4 and 9.0 nM for TEM-1 and SHV-1 (class A), respectively, and 4.8 nM in AmpC (class C) beta-lactamases. The compound also inhibits irreversibly the class C extended-spectrum GC1 beta-lactamase (IC(50) = 6.2 nM). High-resolution crystallographic structures of a reaction intermediate of (5R)-(6Z)-6-(5,6-dihydro-8H-imidazo[2,1-c][1,4]oxazin-2-ylmethylene)-7-oxo-4-thia-1-azabicyclo[3.2.0]hept-2-ene-3-carboxylic acid 1 with the SHV-1 beta-lactamase and with the GC1 beta-lactamase have been determined by X-ray diffraction to resolutions of 1.10 and 1.38 A, respectively. The two complexes were refined to crystallographic R-factors (R(free)) of 0.141 (0.186) and 0.138 (0.202), respectively. Cryoquenching of the reaction of 1 with each beta-lactamase crystal produced a common, covalently bound intermediate. After acylation of the serine, a nucleophilic attack by the departing thiolate on the C6' atom yielded a novel seven-membered 1,4-thiazepine ring having R stereochemistry at the new C7 moiety. The orientation of this ring in each complex differs by a 180 degrees rotation about the bond to the acylated serine. The acyl ester bond is stabilized to hydrolysis through resonance stabilization with the dihydrothiazepine ring and by low occupancy or disorder of hydrolytic water molecules. In the class A complex, the buried water molecule on the alpha-face of the ester bond appears to be loosely bound or absent. In the class C complex, a water molecule on the beta-face is disordered and poorly activated for hydrolysis. Here, the acyl intermediate is unable to assist its own hydrolysis, as is thought to occur with many class C substrates.     

Effects of N-alkyl and ammonium groups on the hydrolytic cleavage of DNA with a Cu(II)TACH (1,3,5-triaminocyclohexane) complex. Speciation, kinetic, and DNA-binding studies for reaction mechanism

cis,cis-1,3,5-Triaminocyclohexane (c-TACH), its N-alkyl-derivatives (alkyl = methyl, ethyl), and trans,cis-1,3,5-triaminocyclohexane (t-TACH) were prepared, and speciation and DNA cleaving property of Cu(II) complexes of these ligands were investigated. All of the complexes efficiently promote the hydrolytic cleavage of supercoiled plasmid DNA under physiological conditions without further additives. The DNA cleavage rate (V(obs)) trend at pH values between 8 and 9 is N-Me(3) = N-Et(1) < t-TACH < c-TACH < N-Et(2) < N-Et(3). At pH 7, the trend is c-TACH < N-Et(3) = N-Et(2) < N-Et(1) < N-Me(3) < t-TACH. The cleavage rate constants at 35 degrees C, for the c-TACH complex are 3 x 10(-1) h(-1) at pH 8.1 and 2 x 10(-1) h(-1) at pH 7.0 ([DNA] = 7 microM, [Cu(II)-complex] = 105 microM). The hydrolytically active species at pH > 8 is CuL(H(2)O)(OH)(+) in which L coordinates to Cu(II) as a tridentate ligand for all complexes except for t-TACH. The hydrolytically active species at pH 7 is CuLH(H(2)O)(3)(3+) or CuLH(H(2)O)(4)(3+) in which LH coordinates as bidentate ligand. DNA-binding constants of c-TACH and t-TACH complexes are presented and the effects of N-alkyl and ammonium groups are discussed in light of the proposed reaction mechanism.     

Discovery of new chemical leads for selective EP1 receptor antagonists

A series of 4-([2-[alkyl(phenylsulfonyl)amino]phenoxy]methyl)benzoic acids were identified as functional PGE(2) antagonists with selectivity for the EP1 receptor subtype starting from a chemical lead 1, which was found while screening our in-house compound library. Discovery of the optimized analogs 21-23 is presented here and structure-activity relationships (SAR) are also discussed.     

Neurospora mrc1 homologue is involved in replication stability and is required for normal cell growth and chromosome integrity in mus-9 and mus-21 mutants

Stalled replication forks easily collapse and such structures can induce DNA strand breaks or toxic recombination products. Therefore, factors involved in stabilization of replication should be important for genome integrity. In our previous study, loss of both ATM and ATR homologues was shown to cause growth defects and chromosome instability in Neurospora crassa. To elucidate the relationships between these defects and replication instability, we focused on one of the viable replication factors, mrc1. We identified an mrc1 homologue from the N. crassa genome database. The mrc1 disruptant was sensitive to the replication inhibitor hydroxyurea (HU) and delayed restart of the cell cycle from HU treatment. Importantly, HU treatment induced histone H2A phosphorylation in the mrc1 mutant but not in the wild type. Furthermore, the HU-induced H2A phosphorylation was completely dependent on the ATM homologue mus-21, and dysfunction of mus-21 increased HU sensitivity of the mrc1 mutant. These results indicate that Neurospora mrc1 is important for stabilization of replication forks and that loss of mrc1 causes activation of the DNA damage checkpoint. Unexpectedly, loss of mrc1 did not affect cell growth, but the deletion of mrc1 reduced hyphal growth speed and conidia viability in the mus-9 and mus-21 mutants. The mrc1 mus-9 and mrc1 mus-21 double mutants also showed accumulation of micronuclei, which is a typical marker of chromosome instability. These results imply that activation of the checkpoint pathway can protect cells from instability of DNA replication caused by loss of mrc1.     

Comparison of beta-lactamases of classes A and D: 1.5-A crystallographic structure of the class D OXA-1 oxacillinase

The crystallographic structure of the Escherichia coli OXA-1 beta-lactamase has been established at 1.5-A resolution and refined to R = 0.18. The 28.2-kD oxacillinase is a class D serine beta-lactamase that is especially active against the penicillin-type beta-lactams oxacillin and cloxacillin. In contrast to the structures of OXA-2, OXA-10, and OXA-13 belonging to other subclasses, the OXA-1 molecule is monomeric rather than dimeric and represents the subclass characterized by an enlarged Omega loop near the beta-lactam binding site. The 6-residue hydrophilic insertion in this loop cannot interact directly with substrates and, instead, projects into solvent. In this structure at pH 7.5, carboxylation of the conserved Lys 70 in the catalytic site is observed. One oxygen atom of the carboxylate group is hydrogen bonded to Ser 120 and Trp 160. The other oxygen atom is more exposed and hydrogen bonded to the Ogamma of the reactive Ser 67. In the overlay of the class D and class A binding sites, the carboxylate group is displaced ca. 2.6 A from the carboxylate group of Glu 166 of class A enzymes. However, each group is equidistant from the site of the water molecule expected to function in hydrolysis, and which could be activated by the carboxylate group of Lys 70. In this ligand-free OXA-1 structure, no water molecule is seen in this site, so the water molecule must enter after formation of the acyl-Ser 67 intermediate.     

Significant evidence for linkage of mite-sensitive childhood asthma to chromosome 5q31-q33 near the interleukin 12 B locus by a genome-wide search in Japanese families

Childhood-onset asthma is frequently found in association with atopy. Although asthmatic children may develop IgE antibodies against variety of allergens, asthma is associated primarily with allergy to house-dust mites, molds, or other allergens. In this study, we conducted a genome-wide linkage search in 47 Japanese families (197 members) with more than two mite-sensitive atopic asthmatics (65 affected sib-pairs) using 398 markers. Multipoint linkage analysis was carried out for atopic asthma as a qualitative trait using the MAPMAKER/SIB program. We observed significant evidence for linkage with maximum lod scores (MLS) of 4.8 near the interleukin 12 B gene locus on chromosome 5q31-q33. In addition, suggestive evidence on 4q35 with MLS = 2.7 and on 13q11 with MLS = 2.4 was obtained. The other possible linkage regions included 6p22-p21.3 (MLS = 2.1), 12q21-q23 (MLS = 1.9), and 13q14.1-q14.3 (MLS = 2.0). Many of the linkage loci suggested in this study were at or close to those suggested by genome-wide studies for asthma in Caucasian populations. The present study suggests the contribution of the interleukin 12 B gene or nearby gene(s) to mite-sensitive atopic asthma and a considerable number of genetic variants common across Caucasians and Japanese populations contributing to asthma, although the relative importance of various variants may differ between the groups.