Gas-phase removal of biofilms from various surfaces using carbon dioxide aerosols

The present study evaluated the removal of Escherichia coli XL1-blue biofilms using periodic jets of carbon dioxide aerosols (a mixture of solid and gaseous CO(2)) with nitrogen gas. The aerosols were generated by the adiabatic expansion of high-pressure CO(2) gas through a nozzle and used to remove air-dried biofilms. The areas of the biofilms were measured from scanning electron micrographs before and after applying the aerosols. The removal efficiency of the aerosol treatment was measured with various air-drying times of the biofilms before the treatment, surface materials, and durations of CO(2) aerosols in each 8-s aerosol-nitrogen cleaning cycle. Nearly 100% of the fresh biofilms were removed from the various surfaces very reliably within 90?s. This technique can be useful for removing unsaturated biofilms on solid surfaces and has potential applications for cleaning bio-contaminated surfaces.     

Purification and characterization of a novel alkaline protease from Bacillus horikoshii

An investigation was conducted on the enhancement of production and purification of an oxidant and SDS-stable alkaline protease (BHAP) secreted by an alkalophilic Bacillus horikoshii, which was screened from the body fluid of a unique Korean polychaeta (Periserrula leucophryna) living in the tidal mud flats of Kwangwha Island in the Korean West Sea. A prominent effect on BHAP production was obtained by adding 2% maltose, 1% sodium citrate, 0.8% NaCl, and 0.6% sodium carbonate to the culturing medium. The optimal medium for BHAP production contained (g/l) SBM, 15; casein, 10; K(2)HPO(4), 2; KH(2)PO(4), 2; maltose, 20; sodium citrate, 10; MgSO(4), 0.06; NaCl, 8; and Na(2)CO(3), 6. A protease yield of approximately 56,000 U/ml was achieved using the optimized medium, which is an increase of approximately 5.5-fold compared with the previous optimization (10,050 U/ml). The BHAP was homogenously purified 34-fold with an overall recovery of 34% and a specific activity of 223,090 U/mg protein using adsorption with Diaion HPA75, hydrophobic interaction chromatography (HIC) on Phenyl-Sepharose, and ion-exchange chromatography on a DEAE- and CMSepharose column. The purified BHAP was determined a homogeneous by SDS-PAGE, with an apparent molecular mass of 28 kDa, and it showed extreme stability towards organic solvents, SDS, and oxidizing agents. The K(m) and k(cat) values were 78.7 μM and 217.4 s(-1) for N-succinyl-Ala- Ala-Pro-Phe-pNA at 37° C and pH 9, respectively. The inhibition profile exhibited by PMSF suggested that the protease from B. horikoshii belongs to the family of serine proteases. The BHAP, which showed high stability against SDS and H(2)O(2), has significance for industrial application, such as additives in detergent and feed industries.     

A protein in crude cytosol regulates glucose-6-phosphatase activity in crude microsomes to regulate group size in Dictyostelium

Dictyostelium discoideum form groups of approximately 2 x 10(4) cells. The group size is regulated in part by a negative feedback pathway mediated by a secreted multipolypeptide complex called counting factor (CF). The CF signal transduction pathway involves CF-repressing internal glucose levels by increasing the K(m) of glucose-6-phosphatase. Little is known about how this enzyme is regulated. Glucose-6-phosphatase is associated with microsomes in both Dictyostelium and mammals. We find that the activity of glucose-6-phosphatase in crude microsomes from cells with high, normal, or low CF activity had a negative correlation with the amount of CF present in these cell lines. In crude cytosols (supernatants from ultracentrifugation of cell lysates), the glucose-6-phosphatase activity had a positive correlation with CF accumulation. The crude cytosols were further fractionated into a fraction containing molecules greater than 10 kDa (S>10K) and molecules less than 10 KDa (S<10K). S>10K from wild-type cells strongly repressed the activity of glucose-6-phosphatase in wild-type microsomes, whereas S>10K from countin(-) cells (cells with low CF activity) significantly increased the activity of glucose-6-phosphatase in wild-type microsomes by decreasing K(m). The regulatory activities in the wild-type and countin(-) S>10Ks are heat-labile and protease-sensitive, suggesting that they are proteins. S<10K from both wild-type and countin(-) cells did not significantly change glucose-6-phosphatase activity. Together, the data suggest that, as a part of a pathway modulating multicellular group size, CF regulates one or more proteins greater than 10 KDa in crude cytosol that affect microsome-associated glucose-6-phosphatase activity.     

Identification and characterization of a novel human collectin CL-K1

Collectins are a family of C-type lectins with two characteristic structures, collagen like domains and carbohydrate recognition domains. They recognize carbohydrate antigens on microorganisms and act as host-defense. Here we report the cloning and characterization of a novel collectin CL-K1. RT-PCR analyses showed CL-K1 mRNA is present in all organs. The deduced amino acid sequence and the data from immunostaining of CL-K1 cDNA expressing CHO cells revealed that CL-K1 is expressed as a secreted protein. CL-K1 is found in blood by immunoblotting and partial amino acid analyses. CL-K1 showed Ca(2+)-dependent sugar binding activity of fucose and weakly mannose but not N-acetyl-galactosamine, N-acetyl-glucosamine, or maltose, though mannose-binding lectin (MBL) containing similar amino acid motif. CL-K1 can recognize specially several bacterial saccharides due to specific sugar-binding character. Elucidation of the role of two ancestor collectins of CL-K1 and CL-L1 could lead to see the biological function of collectin family.     

Synthesis of 2-C-hydroxymethylribofuranosylpurines as potent anti-hepatitis C virus (HCV) agents

On the basis of potent anti-HCV activity of 2'-C-methyladenosine, novel 2'-C-hydroxymethyladenosine analogues 2a-c were synthesized from d-ribose in order to lead to favorable interaction with HCV polymerase. Among compounds tested, adenosine derivative 2a exhibited potent anti-HCV activity, indicating that the hydroxyl group of 2'-C-hydroxymethyl substituent led to favorable interaction with HCV polymerase.     

Theoretical investigation of the photoinitiated folding of HP-36

A computational model was developed to examine the phototriggered folding of a caged protein, a protein modified with an organic photolabile cross-linker. Molecular dynamics simulations of the modified 36-residue fragment of subdomain B of chicken villin head piece with a photolabile linker were performed, starting from both the caged and the uncaged structures. Construction of a free-energy landscape, based on principal components as well as on radius of gyration versus root-mean-square deviation, and circular dichroism calculations were employed to characterize folding behavior and structures. The folded structures observed in the molecular dynamics trajectories were found to be similar to that of the wild-type protein, in agreement with the published experimental results. The free-energy landscapes of the modified and wild-type proteins have similar topology, suggesting common thermodynamic/kinetic behavior. The existence of small differences in the free-energy surface of the modified protein from that of the native protein, however, indicates subtle differences in the folding behavior.     

Mutual Regulation of ntcA and hetR during Heterocyst Differentiation Requires Two Similar PP2C-Type Protein Phosphatases,PrpJ1 and PrpJ2, in Anabaena sp. Strain PCC 7120

The filamentous cyanobacterium Anabaena sp. strain PCC 7120 can form heterocysts for N₂ fixation. Initiation of heterocyst differentiation depends on mutual regulation of ntcA and hetR. Control of hetR expression by NtcA is partially mediated by nrrA, but other factors must be involved in this regulation. Anabaena has two closely related PP2C-type protein phosphatases, PrpJ1 (formerly PrpJ) and PrpJ2; PrpJ1 is involved in heterocyst maturation. In this study, we show that PrpJ2, like PrpJ1, has Mn²⁺-dependent phosphatase activity. We further demonstrate that whereas prpJ2 is dispensable for cell growth under different nitrogen regimens tested, a double mutant with both prpJ1 and prpJ2 disrupted did not initiate heterocyst differentiation. Ectopic expression of hetR in the double mutant could rescue the failure to initiate heterocyst development, but the heterocysts formed, like those of a prpJ1 single mutant, were not mature. The expression of prpJ2 was enhanced during heterocyst development, and the upregulation of the gene was directly under the control of NtcA. Upregulation of both ntcA and hetR was affected in the double mutant. We propose that PrpJ1 and PrpJ2 together are required for mutual regulation of ntcA and hetR and are thus involved in regulation of the initiation of heterocyst differentiation.Many cyanobacteria can fix N₂ when combined nitrogen sources become limiting in the growth medium. The nitrogenase enzymatic complex responsible for nitrogen fixation is very sensitive to oxygen, and oxygen is produced by photosynthesis by cyanobacteria. The strategy used by some filamentous diazotrophic cyanobacteria to resolve this oxygen paradox is to perform photosynthesis and nitrogen fixation in two distinct cell types, differentiated cells called heterocysts that provide a microoxic environment for nitrogenase and vegetative cells which perform oxygenic photosynthesis (22, 36, 39). One such organism is Anabaena sp. strain PCC 7120. In this strain, heterocysts account for 5 to 10% of the cells and appear in a semiregular pattern along each filament. Therefore, the process of heterocyst differentiation provides a prokaryotic model to study developmental pattern formation. Three factors account for the microoxic environment in heterocysts: the heterocyst envelope composed of an inner layer of glycolipid surrounded by an outer layer of polysaccharides that limits oxygen penetration, the lack of oxygen-producing photosystem II, and an increased rate of respiration to consume oxygen (36).The initiation of heterocyst differentiation and the formation of the heterocyst pattern are governed by multiple signals and the concerted actions of several proteins as positive or negative regulators (for a recent review, see 39). The accumulation of 2-oxoglutarate following limitation of combined nitrogen is a trigger that initiates heterocyst development by stimulating the DNA-binding activity of NtcA, a protein involved in the regulation of carbon and nitrogen metabolism, as well as initiation of heterocyst differentiation (7, 12, 13, 18, 20, 32, 35). HetR, a protease with DNA-binding activity, plays a central role in the early steps of heterocyst differentiation (14, 40). Both ntcA and hetR are autoregulated, and the expression of hetR and the expression of ntcA are mutually dependent because upregulation of one of theses genes is dependent on the other gene (3, 4, 23). How HetR regulates the expression of ntcA remains unknown. No NtcA-binding site has been found in the upstream region of hetR, and the regulation of hetR by NtcA could be partially due to the action of the response regulator NrrA (8, 9, 24). However, NrrA cannot be the only link between ntcA and hetR, because when nrrA was inactivated, both heterocyst differentiation and hetR upregulation were only delayed (8). Indeed, ccbP, encoding a calcium-binding protein, is regulated by NtcA, and it has been proposed that the pool of calcium affects the activity of HetR (31).The genome of Anabaena sp. strain PCC 7120 contains a large number of genes encoding two-component signaling systems, protein Ser/Thr and/or Tyr kinases, and phosphatases, including eight genes encoding PP2C-type Ser/Thr phosphatases (16, 26, 34, 38). Some of these genes are involved in heterocyst development, mostly in heterocyst maturation and functioning (8, 11, 17, 19, 21, 25, 30, 37). We have shown previously that PrpJ is a PP2C-type protein phosphatase located on the plasma membrane (15). A prpJ1 mutant (strain S20) failed to grow under diazotrophic conditions and formed heterocysts lacking the major heterocyst-specific glycolipid (HGL), in contrast to other mutants whose mutations affect either the synthesis or the deposition of both the major and minor HGLs (1, 2, 10, 28) or only the minor HGL (30). Therefore, PrpJ represents a new regulatory branch for heterocyst maturation, possibly involving regulation of only a subset of genes involved in glycolipid synthesis. These observations indicate that multiple input pathways participate in the maturation of heterocysts. When proheterocysts were formed, filaments of the prpJ1 mutant, fragmented extensively at the junctions between proheterocysts and vegetative cells, resulting in free nonmature heterocysts and filaments that were 11 cells long on average (15).Open reading frame all2470 encodes one member of the PP2C family of protein phosphatases in Anabaena sp. strain PCC 7120 (35). The deduced amino acid sequence of All2470 is similar to that of PrpJ, and these two proteins have similar architectures, with an N-terminal domain having an unknown function, a central domain similar to the catalytic domains of PP2C-type protein phosphatases, and a C-terminal domain with a putative transmembrane motif (Fig. ​(Fig.1).1). The amino acid sequences of these two proteins share 40% identity overall, and their catalytic domains are 45% identical. Because these two protein phosphatases are very similar, here we use the designations PrpJ1 (formerly PrpJ) for All1731 and PrpJ2 for All2470. In the present study, we show that PrpJ1 and PrpJ2 are involved in the initiation of heterocyst differentiation by acting on the mutual regulation of ntcA and hetR.Open in a separate windowFIG. 1.(A) Different domains of PrpJ1 and PrpJ2. The length of each domain (in number of residues) is indicated in parentheses. TM, putative transmembrane domain. (B) Genomic environment of prpJ2 and strategy for inactivating prpJ2 by insertion of an antibiotic resistance cassette (Neo^r). The arrow for the Neo^r cassette indicates the orientation of the resistance cassette relative to that of prpJ2.