Roles of focal adhesions and fibronectin‐mediated cohesion in proliferation of confluent fibroblasts

Multilayered fibroblast sheets have applications as cell transplants for tissue engineering. One way to increase their therapeutic efficacy is to increase cell numbers in a graft, but the factors influencing multilayered growth remain poorly understood. In this study, we investigated the roles of focal adhesion (FA) assembly and intercellular cohesion through fibronectin (FN) in the proliferation of normal human fibroblasts at confluence. Density‐dependent growth‐arrested fibroblasts resumed DNA synthesis when cultured in multilayer formation medium (MFM) containing transforming growth factor‐β1, ascorbic acid, and serum. This proliferation depended on α5β1‐integrin‐mediated cell‐FN‐cell interactions because blocking them with antibodies inhibited DNA synthesis. However, cell‐FN‐cell cohesion operated well regardless of exposure to MFM, judging from several parameters, including FN matrix deposition, activated β1 integrin expression, and stress fiber development. Density‐arrested cells formed few FAs at the cell center. Exposure of the cells to MFM induced the formation of vinculin‐, paxillin‐, and phosphotyrosine‐containing FAs throughout the ventral cell‐surface, indicating ROCK‐mediated actomyosin contractile force generation. When the assembly of FAs was inhibited with either the ROCK inhibitor Y‐27632 or the myosin II inhibitor blebbistatin, the up‐regulation of DNA synthesis by MFM was suppressed. The drugs did not impair FN matrix deposition, activated β1 integrin expression, and stress fiber development. Thus, these results indicate that the formation of FAs promotes the proliferation of confluent fibroblasts with the support of α5β1‐integrin‐mediated cell‐FN‐cell cohesion. The present findings provide insights into the rational design of high‐density fibroblast transplants. J. Cell. Physiol. 219: 194–201, 2009. © 2008 Wiley‐Liss, Inc.     

Taxonomy of Clostridium tetani and related species.

Clostridium tetani and its related species C. tetanomorphum, C. cochlearium and C. lentoputrescens were examined for DNA-DNA homology and biochemical properties. Two distinctly different groups were included under the name of C. tetanomorphum: one was identical with C. cochlearium and the name C. tetanomorphum was applied to the other group with some amendment of biochemical properties. Comparison of the type strain of C. lentoputrescens with wild strains obtained from horse faeces indicated that the name C. lentoputrescens should be abolished as a later synonym of C. cochlearium. Liquefaction of gelatin and spore shape, which have been used as the important criteria for differentiation of C. tetani-related species, were genetically insignificant.     

The chemical modification of beef liver catalase. V. Ethoxyformylation of histidine and tyrosine residues of catalase with diethylpyrocarbonate.

In order to elucidate the possible roles of histidine and tyrosine residues of catalase [EC 1.11.1.6] in maintaining the quaternary structure and catalatic activity, diethylpyrocarbonate modification experiments were carried out. A method for the estimation of N-ethoxyformyl (EF)-His at pH 5--7 and of O-ethoxyformyl (EF)-Tyr in alkaline solution by measuring A 242 nm (ximM = 3.2) and A278 nm (ximM = 1.16), respectively, was developed. The formation of EF-His and EF-Tyr was an electrophilic reaction and was dependent on pH, exhibiting pK values of 6.8 and 9.9, respectively. The maximal yield of EF-His at pH 6.0 was 49% of the total histidine content, but no inactivation nor unfolding of the enzyme was observed. The formation of 12 EF-Tyr residues per mole of catalase at pH 8.1 did not cause any inactivation, but the formation of 8 more EF-Tyr residues at pH 8.9 resulted in both inactivation and unfolding. Nearly complete inactivation and partial splitting of catalase were observed when 43-46 EF-Tyr residues per mole were produced at pH 10.0. More EF-His residues were formed by the reaction of diethyl pyrocarbonate with cyanoethylated (CE)-catalase monomer (subunit) than with CE-catalase tetramer. The CE-catalase tetramer and monomer were extensively O-ethoxyformylated, reaching 100% EF-Tyr formation. These results indicate that a half of the histidine residues may lie outside the protein core and that three-quarters of the tyrosine residues are probably in the protein core of the enzyme. The production of 2--3 EF-Tyr residues per mole of the monomer by ethoxyformylation at pH 7.0 was accompanied by a decrease in the magnitude of the Soret peak. A possible interaction of those tyrosine residues with porphyrin of the heme group is discussed.     

An inhibitor of cyclic AMP-dependent protein kinase enhances the superoxide production of human neutrophils stimulated by N-formyl-methionyl-leucyl-phenylalanine

Intact human neutrophils produced superoxide (O₂ ^–) by the stimulation with N-formyl-methionyl-leucyl-phenylalanine (fMLP) even when the extracellular Ca²⁺ was absent (0.56±0.13 nmol/min per 10⁶ cells). The production by fMLP was enhanced more than twice in the presence of the extracellular Ca²⁺. Moreover, the O₂ ^– production by fMLP in the presence of extracellular Ca²⁺ was enhanced nearly three times by the treatment of cells with H-89, an inhibitor of cyclic AMP-dependent protein kinase (PKA). The enhancement was not observed when the extracellular Ca²⁺ was depleted from the reaction mixture. In addition, H-89 did not enhance fMLP-induced O₂ ^– production of electropermeabilized neutrophils in which the intracellular Ca²⁺ concentration was fixed to about 100 nM. These observations suggest that not only Ca²⁺ influx but the inhibition of PKA is necessary for the maximum O₂ ^– production by fMLP and that the O₂ ^– production is partially suppressed by the activation of PKA induced by fMLP.     

Constructing an improved pyrroloquinoline quinone glucose dehydrogenase binding aptamer for enzyme labeling

Motif analysis of PGa4 indicated that eight consecutive guanine residues were most important for pyrroloquinoline quinine glucose dehydrogenase (PQQGDH) recognition. Because the truncated mutants minimized unexpected interactions between PGa4 and the target-binding aptamer, they will now facilitate the construction of a highly sensitive detection system using a PQQGDH-labeled diagnostic marker-binding aptamer.     

The dominant bacteria shifted from the order “ Lactobacillales ” to Bacillales and Actinomycetales during a start-up period of large-scale, completely-mixed composting reactor using plastic bottle flakes as bulking agent

Bacterial community succession in the start-up of a large-scale, completely-mixed composting reactor was analyzed by 16S rRNA gene (16S rDNA) clone analysis and denaturing gradient gel electrophoresis (DGGE) combined with measurements of temperature, pH, moisture contents, and decomposing rate. DGGE analysis and physicochemical parameters showed that bacterial community succession occurred in four phases; (1) at the start of operation and pH decreasing period (day 0–3), (2) pH decreased and increased period (day 4–11), (3) middle term, moisture content decreasing and maximum temperature increased period (day 12–16) and (4) latter term, temperature decreasing period (day 17–24). Lactobacillus spp. and Bacillus coagulans were detected from the initial phase and middle term, respectively. 16S rDNA clone analysis showed that the dominant bacteria shifted from the order “Lactobacillales” to Bacillales and Actinomycetales. The order “Lactobacillales” was unique which may be caused by using the plastic bottle flakes (polyethylene terephthalate, PET) as bulking agent.     

Improvement of compactin (ML-236B) production by genetic engineering in compactin high-producing <Emphasis Type="Italic">Penicillium citrinum</Emphasis>

An increase in compactin (ML-236B) production was achieved by introducing a whole compactin biosynthetic gene cluster or the regulatory gene mlcR into compactin high-producing Penicillium citrinum. In the previous report, we introduced mlcR encoding the positive regulator of compactin biosynthetic genes into compactin high-producing strain no. 41520, and most of the transformants produced higher amounts of compactin. Here, we characterize one of the resulting high producers (strain TIR-35, which produced 50% more compactin) and reveal that TIR-35 contained five copies of mlcR and that early, enhanced expression of mlcR caused compactin overproduction. Similarly, the introduction of mlcR into strain T48.19, which was created previously from strain no. 41520 by introducing a partial compactin biosynthetic gene cluster, enhanced compactin production further. Our results indicated that genetic engineering is an effective tool to improve compactin production, even in compactin high producers.     

Identification of genes encoding photoconvertible (Class I) water-soluble chlorophyll-binding proteins from Chenopodium ficifolium

Photoconvertible water-soluble chlorophyll-binding proteins, called Class I WSCPs, have been detected in Chenopodiaceae, Amaranthaceae and Polygonaceae plant species. To date, Chenopodium album WSCP (CaWSCP) is the only cloned gene encoding a Class I WSCP. In this study, we identified two cDNAs encoding Chenopodium ficifolium Class I WSCPs, CfWSCP1, and CfWSCP2. Sequence analyses revealed that the open reading frames of CfWSCP1 and CfWSCP2 were 585 and 588 bp, respectively. Furthermore, both CfWSCPs contain cystein2 and cystein30, which are essential for the chlorophyll-binding ability of CaWSCP. Recombinant CfWSCP1 and CfWSCP2, expressed in Escherichia coli as hexa-histidine fusion proteins (CfWSCP1-His and CfWSCP2-His), formed inclusion bodies; however, we were able to solubilize these using a buffer containing 8 M urea and then refold them by dialysis. The refolded CfWSCP1-His and CfWSCP2-His could bind chlorophylls and exhibited photoconvertibility, confirming that the cloned CfWSCPs are further examples of Class I WSCPs.