The plasmid F OmpP protease, a homologue of OmpT, as a potential obstacle to E. coli-based protein production

OmpT, an outer membrane-localized protease of Escherichia coli, cleaves a number of exogenous and endogenous proteins during their purification. SecY, an endogenous membrane protein, is a target of this artificial proteolysis in vitro. Here we report that SecY cleavage occurs even in cell extracts from ompT-disrupted cells, if they carry an F plasmid derivative. A gene, ompP, on the F plasmid was shown to be responsible for this proteolysis. These results indicate that the absence of an F-like plasmid should be checked when choosing a host strain for E. coli-based protein production.     

High anti-Helicobacter pylori antibody seropositivity associated with the combination of IL-8-251TT and IL-10-819TT genotypes

Background. Helicobacter pylori induces inflammation of gastric mucosa regulated by several interleukins. This study examined associations between anti‐Helicobacter pylori immunoglobulin G antibody seropositivity and functional polymorphisms of interleukin‐8 T‐251 A and interleukin‐10 T‐819C. Materials and Methods. The subjects were 454 health check‐up examinees (126 males and 328 females) without a history of cancer, aged 35–85 years, residing in Nagoya, Japan. After written informed consent was obtained individually, residual blood was anonymously applied for anti‐Helicobacter pylori immunoglobulin G antibody testing and genotyping by the polymerase chain reaction with confronting two‐pair primers. Results. The genotype frequency of interleukin‐8 T‐251 A was 52.2% for TT, 39.5% for TA, and 8.3% for AA, and that of interleukin‐10 T‐819C was 49.5% for TT, 39.9% for TC and 10.6% for CC. Although the differences in the positive rates among the genotypes were not marked, 115 individuals with interleukin‐8–251TT (low expression genotype) and interleukin‐10–819TT (high expression genotype) had a higher rate (63.5%) than the others (52.0%). Relative to the combination of interleukin‐8–251TT and interleukin‐10–819TT, the sex‐age‐adjusted odds ratio for those with the other combinations was 0.62 (95% confidence interval, 0.39–0.98). The adjusted odds ratio among 65 current smokers was 0.13 (0.03–0.61). Conclusions. The observed association suggests that individuals with interleukin‐8–251TT and interleukin‐10–819TT, a combination presumably causing mild inflammation, have a higher probability of the continuing Helicobacter pylori infection, especially among current smokers.     

Molecular mechanism for the enhancement of arbekacin resistance in a methicillin-resistant Staphylococcus aureus

We have clinically isolated a methicillin-resistant Staphylococcus aureus (MRSA) K-1 which exhibits enhanced arbekacin (Abk) resistance. In this study, we investigated a molecular mechanism for the overproduction of a bifunctional enzyme catalyzing both 2"-O-phosphorylation and 6'-N-acetylation of aminoglycoside antibiotics that is encoded by aacA-aphD and designated [AAC(6')/APH(2")] and is expressed in MRSA K-1. The sequence analysis of the 5'-adjacent region of the aacA-aphD structural gene in MRSA K-1 showed that 12 bp are deleted from the aacA-aphD promoter region when compared with that in MRSA B-26, which exhibits lower resistance to Abk than K-1. By artificially deleting the 12 bp from the corresponding region in MRSA B-26, we confirmed that the strain increases Abk resistance to the same level as seen in MRSA K-1, which suggests that the 12 bp deletion from the 5'-adjacent region of the aacA-aphD structural gene created a strong promoter to overexpress the bifunctional enzyme.     

Phorbol esters alter alpha4 and alphad integrin usage during eosinophil adhesion to VCAM-1

We examined the effect of the protein kinase C activator phorbol-12-myristate-13-acetate (PMA) on the human eosinophil adhesion molecule phenotype and attachment to VCAM-1 via alpha4 and alphad integrins under static and flow conditions. PMA increased surface expression of alphad integrins and decreased alpha4 integrin expression. Under static conditions, eosinophils bound well to VCAM-1, primarily via alpha4beta1 integrins, with a minor alphadbeta2 integrin component. Unexpectedly, PMA-stimulated eosinophils bound equally well to VCAM-1 and albumin in a temperature- and divalent cation-dependent manner, yet adhesion was independent of beta1 and beta2 integrins. Under flow conditions, eosinophils readily attached to VCAM-1, and adhesion was inhibited by both alpha4 and alphad mAbs (95 and 50% inhibition, respectively). Many fewer PMA-stimulated eosinophils bound to VCAM-1 under flow conditions, but both alpha4 and alphad mAbs inhibited adhesion equally. Thus, PMA alters eosinophil integrin expression and the relative contributions of alpha4 and alphad integrins during attachment to VCAM-1.     

Roles of toll-like receptors in C-C chemokine production by renal tubular epithelial cells

Pyelonephritis, in which renal tubular epithelial cells are directly exposed to bacterial component, is a major predisposing cause of renal insufficiency. Although previous studies have suggested C-C chemokines are involved in the pathogenesis, the exact source and mechanisms of the chemokine secretion remain ambiguous. In this study, we evaluated the involvement of Toll-like receptors (TLRs) in C-C chemokine production by mouse primary renal tubular epithelial cells (MTECs). MTECs constitutively expressed mRNA for TLR1, 2, 3, 4, and 6, but not for TLR5 or 9. MTECs also expressed MD-2, CD14, myeloid differentiation factor 88, and Toll receptor-IL-1R domain-containing adapter protein/myeloid differentiation factor 88-adapter-like. Synthetic lipid A and lipoprotein induced monocyte chemoattractant protein 1 (MCP-1) and RANTES production in MTECs, which strictly depend on TLR4 and TLR2, respectively. In contrast, MTECs were refractory to CpG-oligodeoxynucleotide in chemokine production, consistently with the absence of TLR9. LPS-mediated MCP-1 and RANTES production in MTECs was abolished by NF-kappaB inhibition, but unaffected by extracellular signal-regulated kinase inhibition. In LPS-stimulated MTECs, inhibition of c-Jun N-terminal kinase and p38 mitogen-activated protein kinase significantly decreased RANTES, but did not affect MCP-1 mRNA induction. Thus, MTECs have a distinct expression pattern of TLR and secrete C-C chemokines in response to direct stimulation with a set of bacterial components.     

Otx2 is required to respond to signals from anterior neural ridge for forebrain specification

Previous analysis employing chimeric and transgenic rescue experiments has suggested that Otx2 is required in the neuroectoderm for development of the forebrain region. In order to elucidate the precise role of Otx2 in forebrain development, we attempted to generate an allelic series of Otx2 mutations by Flp- and Cre-mediated recombination for the production of conditional knock-out mice. Unexpectedly, the neo-cassette insertion created a hypomorphic Otx2 allele; consequently, the phenotype of compound mutant embryos carrying both a hypomorphic and a null allele (Otx2(frt-neo/-)) was analyzed. Otx2(frt-neo/-) mutant mice died at birth, displaying rostral head malformations. Molecular marker analysis demonstrated that Otx2(frt-neo/-) mutant embryos appeared to undergo anterior-posterior axis generation and induction of anterior neuroectoderm normally; however, these mutants subsequently failed to correctly specify the forebrain region. As the rostral margin of the neural plate, termed the anterior neural ridge (ANR), plays crucial roles with respect to neural plate specification, we examined expression of molecular markers for the ANR and the neural plate; moreover, neural plate explant studies were performed. Analyses revealed that telencephalic gene expression did not occur in mutant embryos due to defects of the neural plate; however, the mutant ANR bore normal induction activity on gene expression. These results further suggest that Otx2 dosage may be crucial in the neural plate with respect to response to inductive signals primarily from the ANR for forebrain specification.     

In situ expression of heat shock proteins,Hsc73, Hsj2 and Hsp86 in the developing tooth germ of mouse lower first molar

This study examined the detailed gene expression pattern of three different heat shock proteins (HSPs), Hsc73, Hsj2, and Hsp86, by means of an in situ hybridization method. Hsc73, Hsj2, and Hsp86 were shown in our previous study to be differentially expressed in the mouse embryonic mandible at day 10.5 (E10.5) gestational age. These HSP genes showed similar expression patterns during development of the mouse lower first molar. HSPs-expressing cells were widely distributed in both the epithelial and underlying ectomesenchymal cells at E10.5, and then were slightly localized at E12 in an area where the tooth germ of the lower first molar is estimated to be formed. A strong expression of HSPs was observed in the tooth germ at E13.5. At the cap stage, HSPs were expressed in the enamel organ and dental papilla. At the bell stage, HSPs were distinctly expressed in the inner enamel epithelium and dental papilla cells facing the inner enamel epithelial layer, which later differentiate into ameloblasts and odontoblasts, respectively. This study is the first report in which Hsc73, Hsj2, and Hsp86 were distinctly expressed in the developing tooth germ, thus suggesting these HSPs are related to the development and differentiation of odontogenic cells.     

ATP binding/hydrolysis by and phosphorylation of peroxisomal ATP-binding cassette proteins PMP70 (ABCD3) and adrenoleukodystrophy protein (ABCD1)

The 70-kDa peroxisomal membrane protein (PMP70) and adrenoleukodystrophy protein (ALDP), half-size ATP-binding cassette transporters, are involved in metabolic transport of long and very long chain fatty acids into peroxisomes. We examined the interaction of peroxisomal ATP-binding cassette transporters with ATP using rat liver peroxisomes. PMP70 was photoaffinity-labeled at similar efficiencies with 8-azido-[alpha-32P]ATP and 8-azido-[gamma-32P]ATP when peroxisomes were incubated with these nucleotides at 37 degrees C in the absence Mg2+ and exposed to UV light without removing unbound nucleotides. The photoaffinity-labeled PMP70 and ALDP were co-immunoprecipitated together with other peroxisomal proteins, which also showed tight ATP binding properties. Addition of Mg2+ reduced the photoaffinity labeling of PMP70 with 8-azido-[gamma-32P]ATP by 70%, whereas it reduced photoaffinity labeling with 8-azido-[alpha-32P]ATP by only 20%. However, two-thirds of nucleotide (probably ADP) was dissociated during removal of unbound nucleotides. These results suggest that ATP binds to PMP70 tightly in the absence of Mg2+, the bound ATP is hydrolyzed to ADP in the presence of Mg2+, and the produced ADP is dissociated from PMP70, which allows ATP hydrolysis turnover. Properties of photoaffinity labeling of ALDP were essentially similar to those of PMP70. Vanadate-induced nucleotide trapping in PMP70 and ALDP was not observed. PMP70 and ALDP were also phosphorylated at a tyrosine residue(s). ATP binding/hydrolysis by and phosphorylation of PMP70 and ALDP are involved in the regulation of fatty acid transport into peroxisomes.     

Fractionation and anti-tumor activity of the mycelia of liquid-cultured Phellinus linteus

All the fractions of Phellinus linteus mycelia showed anti-tumor activity toward solid tumors planted in mice. The highest anti-tumor activity of 81.2% was observed in the protein-glucan complex obtained by precipitating the 24% NaOH extract at pH 6.0. This protein-glucan complex consisted of 39.3% polysaccharide and 49.4% protein. Its (13)C- and (1)H-NMR data showed that the main glucan part of the complex was simple alpha-1,3-glucan chains.