Claudin-5-Binders Enhance Permeation of Solutes across the Blood-Brain Barrier in a Mammalian Model.

A current bottleneck in the development of central nervous system (CNS) drugs is the lack of drug delivery systems targeting the CNS. The intercellular space between endothelial cells of the blood-brain barrier (BBB) is sealed by complex protein-based structures called tight junctions (TJs). Claudin-5 (CLDN-5), a tetra-transmembrane protein is a key component of the TJ seal that prevents the paracellular diffusion of drugs into the CNS. In the present study, to investigate whether CLDN-5 binders can be used for delivery of drugs to the CNS, we generated monoclonal antibodies (mAbs) specific to the extracellular domains of CLDN-5. In an in vitro model of the BBB, the anti-CLDN-5 mAbs attenuated trans-epithelial/endothelial electrical resistance and enhanced solute permeation. These anti-CLDN-5 mAbs are potential leads for the development of novel drug delivery systems targeting the CNS.

Monoclonal antibodies against extracellular domains of claudin-1 block hepatitis C virus infection in a mouse model.

UNLABELLED: Hepatitis C virus (HCV) entry into host cells is a complex process requiring multiple host factors, including claudin-1 (CLDN1). Safe and effective therapeutic entry inhibitors need to be developed. We isolated a human hepatic Huh7.5.1-derived cell mutant that is nonpermissive to HCV, and comparative microarray analysis showed that the mutant was CLDN1 defective. Four hybridomas were obtained, which produced monoclonal antibodies (MAbs) that interacted with the parental Huh7.5.1 cell but not with the CLDN1-defective mutant. All MAbs produced by these hybridomas specifically bound to human CLDN1 with a very high affinity and prevented HCV infection of Huh7.5.1 cells in a dose-dependent manner, without apparent cytotoxicity. Two selected MAbs also inhibited HCV infection of human liver-chimeric mice without significant adverse effects. CLDN1 may be a potential target to prevent HCV infection in vivo. Anti-CLDN1 MAbs may hence be promising candidates as novel anti-HCV agents. IMPORTANCE: Safe and effective therapeutic entry inhibitors against hepatitis C virus (HCV) are very useful for combination therapies with other anti-HCV drugs, such as direct-acting antivirals. In this study, we first showed an effective strategy for developing functional monoclonal antibodies (MAbs) against extracellular domains of a multimembrane-spanning target protein, claudin-1 (CLDN1), by using parental cells expressing the intact target membrane protein and target-defective cells. The established MAbs against CLDN1, which had a very high affinity for intact CLDN1, efficiently inhibited in vitro and in vivo HCV infections. These anti-CLDN1 MAbs are promising leads for novel entry inhibitors against HCV.

Improvement of pustulosis palmaris et plantaris by periodontal infection control in a patient with chronic periodontitis.

BACKGROUND: Pustulosis palmaris et plantaris (PPP) is a chronic, inflammatory, autoimmune-type disease characterized by sterile pustules of skin. The skin inflammation is influenced by several factors such as drugs, sunlight, metabolic and psychogenic factors as well as metal allergy. Here, we report a rare case that intensive periodontal treatment might have contributed to the improvement of skin inflammation. RESULTS: Skin inflammation regressed 1 month after intensive periodontal treatment. Both CD4/CD8 ratio and % of B cells in the blood sample were slightly decreased corresponding to the improvement of periodontal inflammation. CONCLUSIONS: Infection control of periodontal lesions might be one of attractive therapeutic targets in management of PPP.

Cell-free protein synthesis system prepared from insect cells by freeze-thawing.

We established a novel cell-free protein synthesis system derived from Trichoplusia ni (HighFive) insect cells by a simple extraction method. Luciferase and beta-galactosidase were synthesized in this system with active forms. We analyzed and optimized (1) the preparation method of the insect cell extract, (2) the concentration of the reaction components, and (3) the 5'-untranslated region (5'-UTR) of mRNA. The extract was prepared by freeze-thawing insect cells suspended in the extraction buffer. This preparation method was a simple and superior method compared with the conventional method using a Dounce homogenizer. Furthermore, protein synthesis efficiency was improved by the addition of 20% (v/v) glycerol to the extraction buffer. Concentrations of the reaction components were optimized to increase protein synthesis efficiency. Moreover, mRNAs containing 5'-UTRs derived from baculovirus polyhedrin genes showed high protein synthesis activity. Especially, the leader composition of the Ectropis obliqua nucleopolyhedrovirus polyhedrin gene showed the highest enhancement activity among the six 5'-UTRs tested. As a result, in a batch reaction approximately 71 microg of luciferase was synthesized per milliliter of reaction volume at 25 degrees C for 6 h. Moreover, this method for the establishment of a cell-free system was applied also to Spodoptera frugiperda 21 (Sf21) insect cells. After optimizing the concentrations of the reaction components and the 5'-UTR of mRNA, approximately 45 microg/mL of luciferase was synthesized in an Sf21 cell-free system at 25 degrees C for 3 h. These productivities were sufficient to perform gene expression analyses. Thus, these cell-free systems may be a useful tool for simple synthesis in post-genomic studies as a novel protein production method.

A thermostable laccase from Streptomyces lavendulae REN-7: purification, characterization, nucleotide sequence, and expression.

We found a polyphenoloxidase (PPO) in the cell extract of Streptomyces lavendulae REN-7. About 0.8 mg of purified PPO was obtained from 200 g of the mycelia with a yield of 9.0%. REN-7-PPO showed broad substrate specificity toward various aromatic compounds. Moreover, this enzyme was capable of oxidation of syringaldazine, which is a specific substrate for laccase. Interestingly, REN-7-PPO retained its original activity after 20 min of incubation at even 70 degrees C. The gene encoding the PPO was cloned. Four copper-binding sites characteristics of laccases were contained in the deduced amino acid sequence. We constructed a high-level expression system of this gene in Escherichia coli. The properties of the recombinant enzyme were identical that of wild-type. In conclusion, this PPO is a thermostable laccase.

Enzymological characterization of EpoA, a laccase-like phenol oxidase produced by Streptomyces griseus.

Laccase is an enzyme that catalyzes the oxidation of phenolic compounds by coupling the reduction of oxygen to water. While many laccases have been identified in plant and fungal species, enzymes of prokaryotic origin are poorly known. Here we report the enzymological characterization of EpoA, a laccase-like extracytoplasmic phenol oxidase produced by Streptomyces griseus. EpoA was expressed and purified with an Escherichia coli host-vector system as a recombinant protein fused with a C-terminal histidine-tag (rEpoA). Physicochemical analyses showed that rEpoA comprises a stable homotrimer containing all three types of copper (types 1-3). Various known laccase substrates were oxidized by rEpoA, while neither syringaldazine nor guaiacol served as substrates. Among the substrates examined, rEpoA most effectively oxidized N,N-dimethyl-p-phenylenediamine sulphate with a Km value of 0.42 mM. Several metal chelators caused marked inhibition of rEpoA activity, implying the presence of a metal center essential for the oxidase activity. The pH and temperature optima of rEpoA were 6.5 and 40 degrees C, respectively. The enzyme retained 40% activity after preincubation at 70 degrees C for 60 min. EpoA-like activities were detected in cell extracts of 8/40 environmental actinomycetes strains, which suggests that similar oxidases are widely distributed among this group of bacteria.

A novel extracytoplasmic phenol oxidase of Streptomyces: its possible involvement in the onset of morphogenesis.

Exogenous addition of copper stimulates cellular differentiation in Streptomyces spp. Several lines of evidence suggested a parallel correlation between the stimulatory effect of copper and phenol-oxidizing enzyme activities in Streptomyces griseus. Here a novel extracytoplasmic phenol oxidase (EpoA) associated with cellular development of this organism was identified and characterized. EpoA activity, examined by an in-gel stain procedure with N,N'-dimethyl-p-phenylenediamine sulfate as a substrate, was repressed by glucose and induced by copper supplied in the medium. The enzyme activity was abolished and markedly reduced in the mutants forA-factor biosynthesis and amfR, respectively, which suggested that the activity of the enzyme depends on those essential regulators for morphogenesis in S. griseus. EpoA protein was purified to homogeneity and the N-terminal amino acid sequence was determined. A homologous sequence identified in the genomic database of Streptomyces coelicolorA3(2) was used as a probe to clone the complete epoA gene of S. griseus. The deduced amino acid sequence of EpoA revealed that the mature protein with a molecular mass of 34 kDa was preceded by a signal peptide consisting of 34 aa, consistent with EpoA being a secreted enzyme. EpoA was predicted to be a laccase-type oxidase by not only the sequence similarity, but its substrate selectivity, oxidizing not tyrosine but dihydroxyphenylalanine (DOPA) to generate melanin pigment. Introduction of epoA on a plasmid partially restored both the EpoA activity and aerial mycelium productivity in an A-factor-deficient mutant. Exogenous supplementation of a substance synthesized by purified EpoA from DOPA stimulated cellular differentiation in S. griseus and several other species. Ultrafiltration indicated that the molecular mass of the putative stimulant synthesized by EpoA is between 500 and 1000 Da.