Construction and Characteristics of a Recombinant Single- Chain Antibody Fragment against Bacterial Type III Secretion

Pseudomonas aeruginosa, a Gram-negative pathogen, causes life-threatening infections. Lung injury and the development of sepsis depend largely on expression of the virulence genes associated with the type III secretion system of this bacterium. The type III secretion system functions as a molecular syringe to deliver type III secretory toxins directly into the cytosol of eukaryotic cells and also acts to inhibit innate immune mechanisms, thereby preventing bacterial clearance. Antibodies against PcrV, the cap structure in the translocational needle of type III secretory apparatus of P. aeruginosa, block toxin translocation of the type III secretion system. We have been investigating the therapeutic use of a recombinant anti-PcrV single-chain antibody. In this chapter, as a preliminary step toward an antibody-based immunotherapy against bacterial infections, we summarize our experience of constructing a recombinant single-chain antibody (called scFv166), in which the heavy (VH) and light chain (VL) variable regions of the anti-PcrV monoclonal IgG are joined by a flexible peptide linker. The practical methodologies used to make recombinant scFv166 against a bacterial protein component are described in detail

Nonenzymatic kinetic resolution of racemic β-hydroxyalkanephosphonates with a chiral copper catalyst

Kinetic resolution of β-hydroxyalkanephosphonates was efficiently performed by 2-fluorobenzoylation in the presence of copper(II) triflate and (R,R)-Ph-BOX as a catalyst with good s value of up to 21

Slr0967 and Sll0939 induced by the SphR response regulator in Synechocystis sp. PCC 6803 are essential for growth under acid stress conditions

AbstractTwo-component signal transduction is the primary signaling mechanism for global regulation of the cellular response to environmental changes. We used DNA microarray analysis to identify genes that were upregulated by acid stress in the cyanobacterium Synechocystis sp. PCC 6803. Several of these genes may be response regulators that are directly involved in this type of stress response. We constructed deletion mutants for the response regulator genes and compared the growth rates of cells transfected with mutant and wild-type genes in a low pH medium. Of these mutants, deletion of sphR affected the growth rate under acid stress (pH 6.0) conditions. We examined genome-wide expression in ΔsphR mutant cells using DNA microarray to determine whether SphR was involved in the regulation of other acid stress responsive genes. Microarray and real-time quantitative reverse-transcription polymerase chain reaction (qRT-PCR) analyses of wild-type cells showed that the expression of phoA, pstS1, and pstS2, which are upregulated under phosphate-limiting conditions, increased (2.48-, 1.88-, and 5.07-fold, respectively) after acid stress treatment for 0.5h. In contrast, pstS2 expression did not increase in the ΔsphR mutant cells after acid stress, whereas the phoA and sphX mRNA levels increased. Furthermore, qRT-PCR and northern blot analysis indicated that downregulation of the acid-responsive genes slr0967 and sll0939 occurred with the deletion of sphR. Indeed, mutants of these genes were more sensitive to acid stress than the wild-type cells. Thus, induction of Slr0967 and Sll0939 by SphR may be essential for growth under acid stress conditions. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial

AtPHT4;4 is a chloroplast-localized ascorbate transporter in Arabidopsis

Ascorbate is an antioxidant and coenzyme for various metabolic reactions in vivo. In plant chloroplasts, high ascorbate levels are required to overcome photoinhibition caused by strong light. However, ascorbate is synthesized in the mitochondria and the molecular mechanisms underlying ascorbate transport into chloroplasts are unknown. Here we show that AtPHT4;4, a member of the phosphate transporter 4 family of Arabidopsis thaliana, functions as an ascorbate transporter. In vitro analysis shows that proteoliposomes containing the purified AtPHT4;4 protein exhibit membrane potential- and Cl-dependent ascorbate uptake. The AtPHT4;4 protein is abundantly expressed in the chloroplast envelope membrane. Knockout of AtPHT4;4 results in decreased levels of the reduced form of ascorbate in the leaves and the heat dissipation process of excessive energy during photosynthesis is compromised. Taken together, these observations indicate that the AtPHT4;4 protein is an ascorbate transporter at the chloroplast envelope membrane, which may be required for tolerance to strong light stress

Transcriptional activation of a hybrid promoter composed of cytomegalovirus enhancer and β-actin/β-globin gene in glomerular epithelial cells in vivo

Transcriptional activation of a hybrid promoter composed of cytomegalovirus enhancer and β-actin/β-globin gene in glomerular epithelial cells in vivo. The aim of this study was to seek a promoter, transactivated selectively in renal cells in vivo by using transgenic (tg) mouse technology. We generated two kinds of tg mouse lines carrying a green fluorescence protein (GFP) cDNA driven either by cytomegalovirus enhancer and β-actin/β-globin promoter (CX-GFP) or by elongation factor la promoter (EF-GFP), and investigated the expression of GFP in the kidney. Microscopic examination of the renal tissues in CX-GFP-tg mice revealed that GFP was expressed only in glomeruli, mainly epithelial cells, but not in tubules, arteries and interstitium. Moreover, in situ hybridization demonstrated that GFP mRNA expression was localized in the glomerular cells. In contrast, GFP was not detectable in the kidney in any of the lines of EF-GFP-tg mouse. To exclude the possible involvement of the GFP cDNA as an enhancer, we constructed tg mice carrying the CX promoter driving a human CD4 cDNA. It was confirmed that the expression patterns of human CD4 in the kidney were quite similar to those of GFP in the kidney of CX-GFP-tg mice. These results strongly suggest that CX promoter could be transactivated in glomerular epithelial cells in vivo

ASK1-dependent recruitment and activation of macrophages induce hair growth in skin wounds

Apoptosis signal-regulating kinase 1 (ASK1) is a member of the mitogen-activated protein 3-kinase family that activates both c-Jun NH2-terminal kinase and p38 pathways in response to inflammatory cytokines and physicochemical stress. We report that ASK1 deficiency in mice results in dramatic retardation of wounding-induced hair regrowth in skin. Oligonucleotide microarray analysis revealed that expression of several chemotactic and activating factors for macrophages, as well as several macrophage-specific marker genes, was reduced in the skin wound area of ASK1-deficient mice. Intracutaneous transplantation of cytokine-activated bone marrow-derived macrophages strongly induced hair growth in both wild-type and ASK1-deficient mice. These findings indicate that ASK1 is required for wounding-induced infiltration and activation of macrophages, which play central roles in inflammation-dependent hair regrowth in skin

Polaprezinc Protects Mice against Endotoxin Shock

Polaprezinc (PZ), a chelate compound consisting of zinc and l-carnosine (Car), is an anti-ulcer drug developed in Japan. In the present study, we investigated whether PZ suppresses mortality, pulmonary inflammation, and plasma nitric oxide (NO) and tumor necrosis factor (TNF)-α levels in endotoxin shock mice after peritoneal injection of lipopolysaccharide (LPS), and how PZ protects against LPS-induced endotoxin shock. PZ pretreatment inhibited the decrease in the survival rate of mice after LPS injection. PZ inhibited the increases in plasma NO as well as TNF-α after LPS. Compatibly, PZ suppressed LPS-induced inducible NO synthase mRNA transcription in the mouse lungs. PZ also improved LPS-induced lung injury. However, PZ did not enhance the induction of heat shock protein (HSP) 70 in the mouse lungs after LPS. Pretreatment of RAW264 cells with PZ suppressed the production of NO and TNF-α after LPS addition. This inhibition likely resulted from the inhibitory effect of PZ on LPS-mediated nuclear factor-κB (NF-κB) activation. Zinc sulfate, but not Car, suppressed NO production after LPS. These results indicate that PZ, in particular its zinc subcomponent, inhibits LPS-induced endotoxin shock via the inhibition of NF-κB activation and subsequent induction of proinflammatory products such as NO and TNF-α, but not HSP induction

Exploration of the Pressurization Condition for Killing Human Skin Cells and Skin Tumor Cells by High Hydrostatic Pressure

High hydrostatic pressure (HHP) is a physical method for inactivating cells or tissues without using chemicals such as detergents. We previously reported that HHP at 200 MPa for 10 min was able to inactivate all cells in skin and giant congenital melanocytic nevus (GCMN) without damaging the extracellular matrix. We also reported that HHP at 150 MPa for 10 min was not sufficient to inactivate them completely, while HHP at 200 MPa for 10 min was able to inactivate them completely. We intend to apply HHP to treat malignant skin tumor as the next step; however, the conditions necessary to kill each kind of cell have not been explored. In this work, we have performed a detailed experimental study on the critical pressure and pressurization time using five kinds of human skin cells and skin tumor cells, including keratinocytes (HEKas), dermal fibroblasts (HDFas), adipose tissue-derived stem cells (ASCs), epidermal melanocytes (HEMa-LPs), and malignant melanoma cells (MMs), using pressures between 150 and 200 MPa. We pressurized cells at 150, 160, 170, 180, or 190 MPa for 1 s, 2 min, and 10 min and evaluated the cellular activity using live/dead staining and proliferation assays. The proliferation assay revealed that HEKas were inactivated at a pressure higher than 150 MPa and a time period longer than 2 min, HDFas and MMs were inactivated at a pressure higher than 160 MPa and for 10 min, and ASCs and HEMa-LPs were inactivated at a pressure higher than 150 MPa and for 10 min. However, some HEMa-LPs were observed alive after HHP at 170 MPa for 10 min, so we concluded that HHP at a pressure higher than 180 MPa for 10 min was able to inactivate five kinds of cells completely

A surgical orthodontic case with multiple tooth losstreated by bite splint with CAD/CAM method

Recently, the number of middle–aged and elderly orthognathic surgery patients with un-healthy oral conditions has been increasing. Although an interdisciplinary approach in-volving orthodontist, oral surgeon and prosthodontist is required in orthognathic surgery of these patients, deciding the jaw position after orthognathic surgery is difficult due to unhealthy oral conditions such as edentulous jaw and tooth loss. In the recently study, preoperative simulation methods for orthognathic surgery have been developed through the use of virtual reality computed technology. A male first examined at the age of 36 years and 7 months was diagnosed as having mandibular protrusion with multiple tooth loss and mandibular deviation. The patient was treatedinterdisciplinary by a team of specialists in surgical orthodontic, dental prosthetic and periodontal treatment. Using the interdisciplin-ary approach and haptic device with virtual tactile perception showed satisfactory results loss treated with orthognathic surgery