Biosurfactant production by Pseudomonas aeruginosa A41 using palm oil as carbon source

Biosurfactant production by Pseudomonas aeruginosa A41, a strain isolated from seawater in the gulf of Thailand, was examined when grown in defined medium containing 2% vegetable oil or fatty acid as a carbon source in the presence of vitamins, trace elements and 0.4% NH(4)NO(3), at pH 7 and 30 degrees C with 200 rpm-shaking for 7 days. The yield of biosurfactant steadily increased even after a stationary phase. Under such conditions the surface tension of the medium was lowered from 55-70 mN/m to 27.8-30 mN/m with every carbon source tested. However, types of carbon sources were found to affect biosurfactant yield. The yields of rhamnolipid biosurfactant were 6.58 g/L, 2.91 g/L and 2.93 g/L determined as rhamnose content when olive oil, palm oil and coconut oil, respectively, were used as a carbon source. Among them, biosurfactant obtained from palm oil was the best in lowering surface tension of the medium. Increase in biosurfactant activities in terms of oil displacement test and rhamnose content were observed to be higher with shorter chain fatty acids than that of the longer chains (C12>C14>C16). In addition, we found that C18:2, highly unsaturated fatty acid, showed higher oil displacement activity and rhamnose content than that of C18:1. The optimal oil displacement activity was found at pH 7-9 and in the presence of 0.5-3% NaCl. The oil displacement activity was stable to temperatures up to 100 degrees C for 15 h. Surface tension reduction activity was relatively stable at pH 2-12 and 0-5% of NaCl. Emusification activity tested with various types of hydrocarbons and vegetable oils showed similarity of up to 60% stability. The partially purified biosurfactant via TLC and silica gel column chromatography gave three main peaks on HPLC with mass spectra of 527, 272, and 661 m/z respectively, corresponding to sodium-monorhamnodecanoate, hydroxyhexadecanoic acid and an unknown compound, respectively.     

Amyloidogenecity and pitrilysin sensitivity of a lysine-free derivative of amyloid beta-peptide cleaved from a recombinant fusion protein

The progressive cerebral deposition of a 40-42 residues amyloid beta-peptide (Abeta) is regarded as a major factor in the onset of the Alzheimer's disease. It has recently been shown that Abeta(1-40) is cleaved by Escherichia coli pitrilysin, a homologue of insulysin, at a specific site. To facilitate the studies on a recognition mechanism of Abeta by pitrilysin, an overproduction system of Abeta(1-40) as a fusion protein with E. coli RNase HI was constructed. This fusion protein was designed such that an Abeta(1-40) derivative, Abeta(1-40)*, in which Lys16 and Lys28 of Abeta(1-40) are simultaneously replaced by Ala, is attached to the C-terminus of E. coli RNase HI and Abeta(1-40)* is separated from RNase HI upon cleavage with lysyl endopeptidase. The fusion protein was overproduced in E. coli in inclusion bodies, solubilized and purified in the presence of guanidine hydrochloride, and cleaved by lysyl endopeptidase. Abeta(1-40)* was purified from the resultant peptide fragments by reverse-phase HPLC. Measurement of the far-UV CD spectra suggests that Abeta(1-40)* is conformationally similar to Abeta(1-40). However, the thioflavin T binding assay suggests that Abeta(1-40)* is more amyloidogenic than Abeta(1-40). Nevertheless, Abeta(1-40)* was cleaved by pitrilysin at the site identical to that in Abeta(1-40).     

Amounts of Sr and Ca Eluted from Deciduous Enamel to Artificial Saliva Related to Dental Caries

This study was performed to elucidate the relationship between dental caries and the levels of Sr and Ca eluted from enamel, and to examine whether these elements are useful as factors to assess caries risk. The available 103 (Sr) and 108 (Ca) samples were obtained among 111 collected deciduous teeth. The healthy regions of enamel were decalcified in artificial saliva at pH 6.2 and 5.5. The eluted levels of these elements from enamel were determined using atomic absorption spectrophotometry. Sr and Ca levels were not affected by the sex nor tooth type. Sr levels of the caries-experienced tooth (CE) group were 2.6-fold (pH 6.2) and 2.2-fold (pH 5.5) higher than those of the sound tooth (ST) group, respectively. Furthermore, the Sr levels were significantly higher in the teeth with treated than in those with untreated caries. Only at pH 6.2 was a significant difference found in Ca levels between the ST and CE groups. In the ST group, at pH 5.5, both the Sr and Ca levels significantly increased when the children had six or more carious teeth. The Sr and Ca elution levels were significantly inhibited in the teeth receiving fluoride application every 3 or 4 months compared to those that were not. These findings indicate that Sr can be an indicator of the acid resistance of teeth, and a useful factor to assess future caries risk.     

NMR and CD analysis of an intermediate state in the thermal unfolding process of mouse lipocalin-type prostaglandin D synthase

We previously reported that the thermal unfolding of mouse lipocalin-type prostaglandin D synthase (L-PGDS) is a completely reversible process under acidic conditions and follows a three-state pathway, including an intermediate state (I) between native state (N) and unfolded state. In the present study, we investigated the intermediate state of mouse C65A L-PGDS and clarified the local conformational changes in the upper and bottom regions by using NMR and CD spectroscopy. The (1)H-(15)N HSQC measurements revealed that the backbone conformation was disrupted in the upper region of the β-barrel at 45°C, which is around the T(m) value for the N ? I transition, but that the signals of the residues located at the bottom region of L-PGDS remained at 54°C, where the maximum accumulation of the intermediate state was found. (1)H-NMR and CD measurements showed that the T(m) values obtained by monitoring Trp54 at the upper region and Trp43 at the bottom region of the β-barrel were 41.4 and 47.5°C, respectively, suggesting that the conformational change in the upper region occurred at a lower temperature than that in the bottom region. These findings demonstrate that the backbone conformation of the bottom region is still maintained in the intermediate state.     

A new prenylflavonoid isolated from propolis collected in the Solomon Islands

The new prenylflavonoid, solophenol A (1), together with three known compounds, bonannione A (2), sophoraflavanone A (3) and (2S)-5,7-dihydroxy-4'-methoxy-8-prenylflavanone (4), were isolated from propolis collected from Malaita Island in The Solomon Islands. The structure of each compound was determined by spectroscopic methods, including mass spectrometry and 2D NMR. Compound 1 exhibited potent 2,2-diphenyl-1-picrylhydrazyl radical-scavenging activity.     

Intraductal polypoid growth variant of pancreatic acinar cell carcinoma metastasizing to the intrahepatic bile duct 6 years after surgery: a case report and literature review

We present the first reported case of intraductal polypoid growth (IPG) variant of pancreatic acinar cell carcinoma (ACC) metastasizing to the intrahepatic bile duct. A 58-year-old Japanese woman had previously presented with obstructive jaundice and a 7.0 cm mass in the pancreatic head. She underwent biliary drainage for 2 months followed by pancreatectomy. Histological examination revealed a carcinoma with acinar pattern, immunohistochemically positive for trypsin, and acinar cell carcinoma was diagnosed. IPGs were prominent in the main pancreatic duct and its tributaries, extending into the intrapancreatic bile duct with tumor casts in the lumen. Imaging examinations 6 years later revealed a growing lesion within the intrahepatic bile duct. Needle biopsy examination suggested metastasis of ACC, and she underwent chemoradiation therapy and partial hepatectomy. Histological examination demonstrated ACC confined to the intrahepatic bile duct. The localization of metastasis and slow growth may indicate indolent biologic behavior of the IPG variant.     

Structure and stability of a thermostable carboxylesterase from the thermoacidophilic archaeon Sulfolobus tokodaii

The hormone-sensitive lipase (HSL) family is comprised of carboxylesterases and lipases with similarity to mammalian HSL. Thermophilic enzymes of this family have a high potential for use in biocatalysis. We prepared and crystallized a carboxylesterase of the HSL family from Sulfolobus?tokodaii (Sto-Est), and determined its structures in the presence and absence of an inhibitor. Sto-Est forms a dimer in solution and the crystal structure suggests the presence of a stable biological dimer. We identified a residue close to the dimer interface, R267, which is conserved in archaeal enzymes of HSL family and is in close proximity to the same residue from the other monomer. Mutations of R267 to Glu, Gly and Lys were conducted and the resultant R267 mutants were characterized and crystallized. The structures of R267E, R267G and R267K are highly similar to that of Sto-Est with only slight differences in atomic coordinates. The dimerized states of R267E and R267G are unstable under denaturing conditions or at high temperature, as shown by a urea-induced dimer dissociation experiment and molecular dynamics simulation. R267E is the most unstable mutant protein, followed by R267G and R267K, as shown by the thermal denaturation curve and optimum temperature for activity. From the data, we discuss the importance of R267 in maintaining the dimer integrity of Sto-Est. Database The atomic coordinates and structural factors have been deposited in the Protein Data Bank with accession numbers of PDB: 3AIK for noninhibited Sto-Est, PDB: 3AIL for DEP-bound, PDB: 3AIM for R267E, PDB: 3AIN for R267G, and PDB: 3AIO for R267K Structured digital abstract ? Sto-Es?and?Sto-Es?bind?by?comigration in gel electrophoresis?(View Interaction:?1,?2) ? Sto-Es?and?Sto-Es?bind?by?x-ray crystallography?(View interaction).     

Requirement of lid2 for interfacial activation of a family I.3 lipase with unique two lid structures

A family I.3 lipase from Pseudomonas sp. MIS38 (PML) is characterized by the presence of two lids (lid1 and lid2) that greatly change conformation upon substrate binding. While lid1 represents the commonly known lid in lipases, lid2 is unique to PML and other family I.3 lipases. To clarify the role of lid2 in PML, a lid2 deletion mutant (ΔL2-PML) was constructed by deleting residues 35-64 of PML. ΔL2-PML requires calcium ions for both lipase and esterase activities as does PML, suggesting that it exhibits activity only when lid1 is fully open and anchored by the catalytically essential calcium ion, as does PML. However, when the enzymatic activity was determined using triacetin, the activity of PML exponentially increased as the substrate concentration reached and increased beyond the critical micellar concentration, while that of ΔL2-PML did not. These results indicate that PML undergoes interfacial activation, while ΔL2-PML does not. The activities of ΔL2-PML for long-chain triglycerides significantly decreased while its activity for fatty acid ethyl esters increased, compared with those of PML. Comparison of the tertiary models of ΔL2-PML in a closed and open conformation, which are optimized by molecular dynamics simulation, with the crystal structures of PML suggests that the hydrophobic surface area provided by lid1 and lid2 in an open conformation is considerably decreased by the deletion of lid2. We propose that the hydrophobic surface area provided by these lids is necessary to hold the micellar substrates firmly to the active site and therefore lid2 is required for interfacial activation of PML. DATABASE: Triacylglycerol lipase (EC 3.1.1.3).     

Construction of three quadruple-fluorescent MDA435 cell lines that enable monitoring of the whole chromosome segregation process in the living state

Mitotic events from prophase to telophase are defined by morphology or movement of chromatin, nuclear envelope, centrosomes and spindles. Live-cell imaging is useful for characterizing the whole chromosome segregation process in the living state. In this study, we constructed three quadruple-fluorescent MDA435 cell lines in which chromatin, kinetochores, nuclear envelope and either inner centromere, microtubules or centrosomes/spindles were differentially visualized with cyan, green, orange and red fluorescent proteins (ECFP, EGFP, mKO and DsRed). Each mitotic stage of the individual cells could be identified by capturing live-cell images without the requirement of fixing or staining steps. In addition, we obtained four-color time-lapse images of one cell line, MDA-Auro/imp/H3/AF, from prophase to metaphase and from early anaphase to telophase. These quadruple-fluorescent cell lines will be useful for precisely analyzing the mitotic events from prophase through to telophase in single cells in the future.     

Crystal structure of highly thermostable glycerol kinase from a hyperthermophilic archaeon in a dimeric form

The crystal structure of glycerol kinase from the hyperthermophilic archaeon Thermococcus kodakaraensis (Tk-GK) in a dimeric form was determined at a resolution of 2.4 A. This is the first crystal structure of a hyperthermophilic glycerol kinase. The overall structure of the Tk-GK dimer is very similar to that of the Escherichia coli glycerol kinase (Ec-GK) dimer. However, two dimers of Ec-GK can associate into a tetramer with a twofold axis, whereas those of Tk-GK cannot. This may be the reason why Tk-GK is not inhibited by fructose 1,6-bisphosphate, because the fructose 1,6-bisphosphate binding site is produced only when a tetrameric structure is formed. Differential scanning calorimetry analyses indicate that Tk-GK is a highly thermostable protein with a melting temperature (T(m)) of 105.4 degrees C for the major transition. This value is higher than that of Ec-GK by 34.1 degrees C. Comparison of the crystal structures of Tk-GK and Ec-GK indicate that there is a marked difference in the number of ion pairs in the alpha16 helix. Four ion pairs, termed IP1-IP4, are formed in this helix in the Tk-GK structure. To examine whether these ion pairs contribute to the stabilization of Tk-GK, four Tk-GK and four Ec-GK derivatives with reciprocal mutations at the IP1-IP4 sites were constructed. The determination of their stabilities indicates that the removal of each ion pair does not affect the stability of Tk-GK significantly, whereas the mutations designed to introduce one of these ion pairs stabilize or destabilize Ec-GK considerably. These results suggest that the ion pairs in the alpha16 helix contribute to the stabilization of Tk-GK in a cooperative manner.