Determination of available breaking stress of agar and gellan gum plate culture methods and the duration of bacterial culture under strong acidic conditions.

AIMS: Several acidophilic bacteria have not been cultured, primarily owing to the lack of suitable culture methods under strong acidic conditions. This study aimed to quantitatively evaluate the strengths of the agar plates (AP) and gellan gum plates (GP), and optimal culture periods under strong acidic conditions. METHODS AND RESULTS: To define the lower limit of plate strength for bacterial isolation culture, the diameter of Escherichia coli K12 colonies and the breaking stress of plates at different concentrations of gelling agents, medium composition and pH conditions were determined. The lower limit of available strength of AP and GP was 19·6 and 14·8 kPa, respectively. Medium composition slightly affected AP breaking stress, although GP with a high cationic concentration medium could not be prepared. CONCLUSIONS: Assessment of the strength limits of AP and GP revealed that AP is not suitable for prolonged bacterial culture (≥72 h). Furthermore, GP was completely ineffective for bacterial culture under highly acidic conditions (≤pH 1·0). SIGNIFICANCE AND IMPACT OF THE STUDY: Our quantitative evaluation method based on breaking stress is a potentially valuable tool to understand the state and the suitable limit of plate culture methods in more detail under various conditions.

Effect of tetrasodium pyrophosphate concentration and cooking time on the physicochemical properties of process cheese.

Tetrasodium pyrophosphate (TSPP) is widely used as an emulsifying salt (ES) in process cheese. Previous reports have indicated that TSPP exhibits some unusual properties, including the gelation of milk proteins at specific ES concentrations. We studied the effect of various concentrations (0.25-2.75%) of TSPP and cooking times (0-20min) on the rheological, textural, and physical properties of pasteurized process Cheddar cheese using a central composite rotatable experimental design. Cheeses were made with a constant pH value to avoid pH as a confounding factor. Modeling of the textural properties of process cheese made with TSPP exhibited complex behavior, with polynomial models (cubic) giving better predictions (higher coefficient of determination values) than simpler quadratic models. Meltability indices (degree of flow from the UW MeltProfiler (University of Wisconsin-Madison), loss tangent value at 60°C from rheological testing, and Schreiber melt area) initially decreased with increasing TSPP concentrations, but above a critical ES concentration (~1.0%) meltability increased at higher TSPP concentrations. The storage modulus values measured at 70°C for process cheese initially increased with increasing TSPP concentration, but above a concentration of 1% ES, the storage modulus values decreased. Cooking time had little effect on the various melting or rheological properties. With an increase in TSPP concentration, the insoluble Ca and P contents increased, suggesting that TSPP addition resulted in the formation of insoluble calcium pyrophosphate complexes; some of which were likely associated with caseins. A portion of the added TSPP remained in the soluble phase. The acid-base buffering profiles also indicated that calcium pyrophosphate complexes were formed in cheese made with TSPP. In milk systems, low levels of TSPP have been shown to induce protein crosslinking and gelation, whereas at higher TSPP concentrations milk gelation was inhibited due to excessive charge repulsion from these calcium pyrophosphate complexes. We hypothesized that a similar phenomenon was occurring in our process cheese, resulting in the initial reduction in meltability with TSPP addition due to protein crosslinking, but at higher TSPP levels meltability increased due to excessive charge repulsion.

RISING beamline (BL28XU) for rechargeable battery analysis.

The newly installed BL28XU beamline at SPring-8 is dedicated to in situ structural and electronic analysis of rechargeable batteries. It supports the time range (1 ms to 100 s) and spatial range (1 µm to 1 mm) needed for battery analysis. Electrochemical apparatus for battery charging and discharging are available in experimental hutches and in a preparation room. Battery analysis can be carried out efficiently and effectively using X-ray diffraction, X-ray absorption fine-structure analysis and hard X-ray photoelectron spectroscopy. Here, the design and performance of the beamline are described, and preliminary results are presented.

Impact of cystic fibrosis transmembrane conductance regulator gene mutation on the occurrence of chronic pancreatitis in Japanese patients.

DNA analyses of the cystic fibrosis transmembrane conductance regulator (CFTR) gene in Japanese patients with idiopathic chronic pancreatitis (ICP) were performed to determine the relationship between the CFTR mutation and ICP. The study included patients with alcoholic pancreatitis (n = 20), patients with ICP (n = 20) and healthy volunteers (controls; n = 110). The poly-T region in intron 8 of the CFTR gene was analysed by direct sequencing. The CFTR coding region was screened using single-strand conformational polymorphism and direct sequencing. In the controls, frequencies of the 5T genotype and 5T allele were 4.5% and 3.6%, respectively. The frequency of the 5T genotype was significantly higher in the ICP group (20%) versus controls, but was not significantly different in alcoholic chronic pancreatitis patients (5%). Thus, the CFTR gene mutation, especially the 5T genotype, appears to have some relationship to ICP prevalence in Japanese patients independent of cystic fibrosis.

UV mutagenesis of Cupriavidus necator for extracellular production of (R)-3-hydroxybutyric acid.

AIM: Ultraviolet (UV) mutagenesis was carried out to obtain mutant strains of Cupriavidus necator that could produce (R)-3-hydroxybutyric acid [(R)-3-HB] in the culture supernatant. METHODS AND RESULTS: C. necator (formerly known as Ralstonia eutropha) was subjected to UV radiation to generate mutants that are capable of producing (R)-3-HB in the culture supernatant. Results indicated that UV mutagen disrupted the phbB (phbB knock-out) and thus, promoted production of (R)-3-HB in mutant strains. Inclusion of acetoacetate esters (carbonyl compounds) in the culture broth led to increased production of (R)-3-HB. Thus, acetoacetyl-CoA (an intermediate of the PHB synthetic pathway) might have been converted to acetoacetate, which in the presence of (R)-3-HB dehydrogenase and NADPH/NADP(+), resulted in extracellular production of (R)-3-HB. CONCLUSIONS: UV mutagenesis proved to be a satisfactory method in generating interesting mutants for extracellular production of (R)-3-HB. Extracellular production of (R)-3-HB upon addition of acetoacetate esters would suggest a likely (R)-3-HB biosynthetic pathway in C. necator. SIGNIFICANCE AND IMPACT OF THE STUDY: Mutants obtained in this study are very useful for production of (R)-3-HB. For the first time, the production of (R)-3-HB by C. necator via acetoacetate is reported.

Development of a novel artificial medium based on utilization of algal photosynthetic metabolites by symbiotic heterotrophs.

AIMS: (i) Quantitative and qualitative analyses of photosynthetic metabolites of Chlorella sorokiniana and elucidation of the mechanism of their utilization by algal symbionts. (ii) Development of artificial medium that imitates photoautotroph-heterotroph interaction and investigation of its suitability for isolation of novel microbes from the environment. METHODS AND RESULTS: Various components, including free dissolved carbohydrates, nitrogenous compounds and vitamin, were detected and together contributed 11.1% (as carbon content) of the total photosynthetic metabolites in the medium. Utilization of these photosynthetic metabolites in algal culture broth by algal symbionts was studied. Many symbionts showed specific utilization patterns. A novel artificial extracellular released organic carbon medium, which imitated the nutritional conditions surrounding algae, was developed based on the pattern of utilization of the algal metabolites by the symbiotic heterotrophs. About 42.9% of the isolates were closely related to photoautotrophic-dependent and oligotrophic bacteria. CONCLUSIONS: With the novel artificial medium, it was possible to selectively isolate some bacterial strains. SIGNIFICANT AND IMPACT OF THE STUDY: Synthetic bacterial growth medium is an important and basic tool for bacterial isolation from environmental samples. The current study shows that preferential separation of typical bacterial subset can be achieved by using artificial medium that mimics photosynthetic metabolites.

Pulmonary exposure to carbon black nanoparticles increases the number of antigen-presenting cells in murine lung.

Particulate matters can enhance antigen-related airway inflammation and immunoglobulin production. The present study was designed to determine the effects of different sizes of nanoparticles on the antigen-presenting cells (APC) in the lung. ICR mice were exposed to vehicle, carbon black (CB) nanoparticles (14 nm or 56 nm), ovalbumin (OVA), or OVA + nanoparticles intratracheally. The expression of major histocompatibility complex (MHC) class II, costimulatory molecules (CD80, CD86, CD11c), and DEC205 (dendritic cell marker), F4/80 (macrophage marker), and CD19 (B-cell marker) in the lung cells was measured by flow cytometry. 14 nm nanoparticles, but not 56 nm nanoparticles, increased the number of the total lung cells. Combination of OVA and 14 nm or 56 nm nanoparticles increased the total lung cells. The expression of MHC class II and/or costimulatory molecules and the number of APC in the lung were increased by 14 nm nanoparticles in the presence or absence of OVA. The increases were more prominent with combination of OVA and 14 nm nanoparticles. 56 nm nanoparticles did not show any significant effects. 14 nm CB nanoparticles can increase the expression of MHC class II and costimulatory molecules and the number of APC in the lung, especially in the presence of antigen, which can result in subsequent antigen-related airway inflammation and immunoglobulin production.

Photobioreactors for mass cultivation of algae.

Algae have attracted much interest for production of foods, bioactive compounds and also for their usefulness in cleaning the environment. In order to grow and tap the potentials of algae, efficient photobioreactors are required. Although a good number of photobioreactors have been proposed, only a few of them can be practically used for mass production of algae. One of the major factors that limits their practical application in algal mass cultures is mass transfer. Thus, a thorough understanding of mass transfer rates in photobioreactors is necessary for efficient operation of mass algal cultures. In this review article, various photobioreactors that are very promising for mass production of algae are discussed.

Fishing for bioactive substances from scorpionfish and some sea urchins.

Venom proteins from the dorsal spine of two scorpionfish, Hypodytes rubripinnis and Synanceia verrucosa were assayed for mitogenicity and cytotoxicity. The two venoms had both mitogenic and cytotoxic activity on murine splenocytes and murine P388 leukemic cells. In H. rubripinnis, the second gel chromatographic fraction showed cytotoxic activity on P388 leukemic cells. On native PAGE, the glycoprotein isolated by concavalin A sepharose chromatography appeared to have a molecular mass of 110 kDa. In addition, two D-galactose-binding lectins (SUL-I and SUL-II) and a heparin-binding lectin (TGL-I) were purified from the globiferous pedicellariae of the toxopneustid sea urchins, Toxopneustes pileolus and Tripneustes gratilla, respectively. SUL-I (Nakagawa et al., 1999a) had mitogenic activity and cytotoxic activity but SUL-II and TGL-I did not. SUL-I did not show sequence homology to SUL-II. A hemolytic lectin with a molecular mass of 29 kDa was isolated from the coelomic fluid of T. gratilla. The hemolytic activity of the lectin was dependent on Ca2+ concentration and inhibited by lactose. The present results suggest that some species of scorpionfish and sea urchins may be novel sources for biologically active substances such as anti-tumor compounds or new lectins.

[Successful non-surgical treatment of acute coronary syndrome involving the left main coronary trunk in four elderly patients].

We encountered 4 patients aged over 80 with acute coronary syndrome involving the left main coronary trunk (LMT) who obtained a successful outcome by non-surgical management. CASE 1: An 80-year-old women suffered acute myocardial infarction. A coronary stent was placed at the orifice of the LMT for dilatation of severe 90% stenosis. Cardiac function was markedly improved after treatment during the chronic period. CASE 2: An 81-year-old man who developed non-sustained ventricular tachycardia. Coronary angiography demonstrated severe stenosis with haziness of the LMT. Intra-aortic balloon pumping was performed for one whole day after the attack and the follow-up study performed one month later revealed that the stenosis was markedly diminished to an insignificant grade without residual ischemia. CASE 3: An 81-year-old man developed acute inferior wall infarction with a background of severe triple vessel disease accompanied by an LMT lesion. Coronary stents were placed at three sites, i.e., the right coronary, LMT, and left anterior descending branch. Though initial treatment was successful, this patient died due to severe arrhythmia. Patients in who CABG is strongly indicated due to LMT lesion complicated with multiple organ disorders will increase as the population of the aged continues to increase in Japan. We obtained satisfactory results by intensive intense non-surgical management including PTCA. From our experience, adequate selection of therapeutic regimens for individual patients is important to improve the long-term prognosis as well as the immediate outcome in the acute stage.

Carbohydrate recognition of gramicidin S analogues in aqueous medium.

We have designed and synthesized of carbohydrate-binding peptides, gramicidin S analogues. Asn/Asp/Gln and Trp residues in the peptides were employed as the binding sites for carbohydrates by hydrogen-bonding interaction and the creation units for hydrophobic pocket to promote the interaction, respectively. The data of fluorescence spectroscopy and affinity column chromatography indicated that the peptides possessed the binding ability for some carbohydrates in aqueous medium. As a result of 1H NMR study, nuclear Overhauser effects between aromatic side chains of a peptide, [Gln(1,1'),Trp(3,3')]-gramisidin S and mannose were observed, indicating that the interaction of the peptide with the sugar occurred in the hydrophobic environment formed by Trp and Phe residues.

Antibacterial activity of bactenecin 5 fragments and their interaction with phospholipid membranes.

Bactenecin 5 (Bac 5) is an antibacterial 43mer peptide isolated from bovine neutrophils. It consists of an Arg-rich N-terminal region and successive repeats of Arg-Pro-Pro-Ile (or Phe). We synthesized Bac 5(1-23) and several related peptides to clarify the roles these regions play in antibacterial activity. An assay of antibacterial activity revealed that such activity requires the presence of Arg residues at or near the N-terminus, as well as a chain length exceeding 15 residues. None of the peptides exhibited haemolytic activity. Polyproline II-like CD curves were observed for most of the peptides. Measurements of the membrane perturbation and fusion indicated that the perturbation and fusogenic activities of the peptides were, generally, parallel to their antibacterial activities. Amino acid substitution in the repeating region had some effect on antibacterial activity.

Impairment of glutathione metabolism in human gastric epithelial cells treated with vacuolating cytotoxin from Helicobacter pylori.

Helicobacter pylori vacuolating cytotoxin (VacA) is believed to be one of the factors that induces gastric disease. Our previous study indicated that VacA causes a decrease in the intracellular ATP level in human gastric epithelial cells, suggesting to impair mitochondrial membrane potential followed by a decrease in energy metabolism (Kimura et al., Microb. Pathog., 1999, 26: 45--52). In the present study, we investigated whether the decrease in ATP level affects glutathione metabolism, in which its synthesis and efflux are ATP-dependent. Treatment of AZ-521 human gastric epithelial cells with 120 nM VacA for 6 h suppressed the efflux of oxidized glutathione (GSSG) in a dose- and time-dependent manner. The efflux of GSSG from the cells and glutathione (GSH) synthesis of cells treated with VacA were approximately 50 and 70% of those of the control, respectively. The turnover rate of intracellular GSH was also suppressed by VacA. Viability of the cells pretreated with VacA, then further incubated with H(2)O(2), was decreased by 50% at 6 h and 70% at 12 h. These results suggested that VacA impairs GSH metabolism in the gastric epithelial cells, which weakens the resistance of the cells against oxidative stress or cellular redox regulation by GSH.

Salmonella enteritidis FliC (flagella filament protein) induces human beta-defensin-2 mRNA production by Caco-2 cells.

Antimicrobial peptides are crucial for host defense at mucosal surfaces. Bacterial factors responsible for induction of human beta-defensin-2 (hBD-2) mRNA expression in Caco-2 human carcinoma cells were determined. Salmonella enteritidis, Salmonella typhimurium, Salmonella typhi, Salmonella dublin, and culture supernatants of these strains induced hBD-2 mRNA expression in Caco-2 human carcinoma cells. Using luciferase as a reporter gene for a approximately 2.1-kilobase pair hBD-2 promoter, the hBD-2-inducing factor in culture supernatant of S. enteritidis was isolated. The supernatant factor was heat-stable and proteinase-sensitive. After purification by anion exchange and gel filtration chromatography, the hBD-2-inducing factor was identified as a 53-kDa monomeric protein with the amino-terminal sequence AQVINTNSLSLLTQNNLNK, which is identical to that of the flagella filament structural protein (FliC) of S. enteritidis. Consistent with this finding, the 53-kDa protein reacted with anti-FliC antibody, which prevented its induction of hBD-2 mRNA in Caco-2 cells. In agreement, the hBD-2-inducing activity in culture supernatant was completely neutralized by anti-FliC antibody. In gel retardation analyses, FliC increased binding of NF-kappaB (p65 homodimer) to hBD-2 gene promoter sequences. We conclude that S. enteritidis FliC induces hBD-2 expression in Caco-2 cells via NF-kappaB activation and thus plays an important role in up-regulation of the innate immune response.

Solution structure of micelle-bound H5 peptide (427-452): a primary structure corresponding to the pore forming region of the voltage dependent potassium channel.

A 26-mer peptide with the sequence of the pore forming region (residues 427-452) of the Shaker K(+) channel (H5 region) was chemically synthesized. Analyses by CD and two-dimensional 1H NMR spectroscopy were used to investigate the structure of the peptide bound to SDS micelles in solution, which are commonly used in biophysical studies. The tertiary structure of the peptide as a monomer was composed of an alpha-helix (431-438), a turn (439-442), and random coils (427-430, 443-452), and was very similar to that of the pore forming region of the native K(+) channel from Streptomyces lividans determined by X-ray analysis. This result suggests that even an isolated peptide forms a native-like conformation for residues from 431 to 442, depending on its intrinsic amino acid sequence and the surrounding environment.

Interaction of mastoparan with membranes studied by 1H-NMR spectroscopy in detergent micelles and by solid-state 2H-NMR and 15N-NMR spectroscopy in oriented lipid bilayers.

Several complementary NMR approaches were used to study the interaction of mastoparan, a 14-residue peptide toxin from wasp venom, with lipid membranes. First, the 3D structure of mastoparan was determined using 1H-NMR spectroscopy in perdeuterated (SDS-d25) micelles. NOESY experiments and distance geometry calculations yielded a straight amphiphilic alpha-helix with high-order parameters, and the chemical shifts of the amide protons showed a characteristic periodicity of 3-4 residues. Secondly, solid-state 2H-NMR spectoscopy was used to describe the binding of mastoparan to lipid bilayers, composed of headgroup-deuterated dimyristoylglycerophosphocholine (DMPC-d4) and dimyristoylphosphatidylglycerol (DMPG). By correlating the deuterium quadrupole splittings of the alpha-segments and beta-segments, it was possible to differentiate the electrostatically induced structural response of the choline headgroup from dynamic effects induced by the peptide. A partial phase separation was observed, leading to a DMPG-rich phase and a DMPG-depleted phase, each containing some mastoparan. Finally, the insertion and orientation of a specifically 15N-labeled mastoparan (at position Ala10) in the bilayer environment was investigated by solid-state 15N-NMR spectroscopy, using macroscopically oriented samples. Two distinct orientational states were observed for the mastoparan helix, namely an in-plane and a trans-membrane alignment. The two populations of 90% in-plane and 10% trans-membrane helices are characterized by a mosaic spread of +/- 30 degrees and +/- 10 degrees, respectively. The biological activity of mastoparan is discussed in terms of a pore-forming model, as the peptide is known to be able to induce nonlamellar phases and facilitate a flip-flop between the monolayers.

Interaction of pleurocidin and its analogs with phospholipid membrane and their antibacterial activity.

A 25-mer cationic peptide pleurocidin, isolated from the winter flounder, has broad antibacterial activity. To clarify the structure-activity relationship, its properties and biological activity were examined. CD measurements showed that pleurocidin took an alpha-helical structure in the presence of DOPC/DOPG (3:1, anionic) vesicles. Very weak hemolytic activity of pleurocidin was observed and its antibacterial activity was moderate. Tryptophan fluorescence shift measurements showed that pleurocidin interacted weakly with a neutral phospholipid, but strongly with an acidic phospholipid. The peptide exhibited weak dye-leakage activity for DOPC (neutral) vesicles and moderate activity for acidic vesicles. From experiments on dye-leakage activity and membrane translocation of the peptide, it seemed likely that pleurocidin, like magainin 2, forms pores in the lipid membrane. A study of amino acid substitution in pleurocidin revealed that alpha-helicity, rather than hydrophobicity, affects the properties and activity of the peptide.

Congenital giant aneurysm of the left atrial appendage mimicking pericardial absence case report.

A 25-year-old man was found to have an abnormal cardiac contour on a chest radiograph, and was referred. Transesophageal echocardiography suggested herniation of the left atrial appendage (LAA) through a gap in the pericardium, and magnetic resonance imaging indicated congenital partial absence of the pericardium. Cardiac dysfunction was caused by compression from the enlarged left atrium and thrombi were thought to be present in the appendage, so surgery was performed. The intraoperative diagnosis was congenital LAA aneurysm. Although distinguishing between congenital LAA aneurysm and congenital absence of the pericardium is reported to be possible with magnetic resonance imaging, we were unable to so in this case.

Efficient production of saikosaponins in Bupleurum falcatum root fragments combined with signal transducers.

An efficient system to produce saikosaponins (saikosaponin-a and -d) in Bupleurum falcatum adventitious root fragments combined with signal transducers was developed. The roots are heterogeneous in terms of size and shape and sometimes form aggregates during cultivation. When the roots were cut to lengths of about 5 mm using a scalpel and cultivated, the root fragments did not form the aggregates, and root growth and saikosaponin production were not inhibited. After screening various signal transducers, it was clear that methyl jasmonate (MeJA) markedly promoted saikosaponin production. By comparing the effect of MeJA and related substances on saikosaponin production, we conclude that both the pentenyl and carboxylmethyl group of MeJA play an important role in the promotion of saikosaponin production. Addition of both 100 microM MeJA and 20 mM CaCl2 to the medium stimulated the content of saikosaponin in the root, with levels reaching 31.7 mg/g-dry root for 15 days of cultivation. A large amount of root fragments were prepared using a blender and cultivated (23 g-dry root/l) with 400 microM MeJA and 20 mM CaCl2, resulting in a high concentration of saikosaponins (747.3 mg/l).

Cationic α-Helical Peptides for Gene Delivery into Cells.

Development of nonviral gene transfer techniques has progressed, particularly the use of several kinds of cationic lipids and cationic polymers such as polylysine derivatives, polyethyleneimines, polyamidoamine dendrimers, and so on, which electrostatically form a complex with the negatively charged DNA, which can be taken up by the cells. Furthermore, targeted gene transfer has also been realized by modification of the gene carriers using cell-targeting ligands such as asialoorosomucoid, transferrin, insulin, or galactose.