Formate-producing capacity provided by reducing ability of Streptococcus thermophilus nicotinamide adenine dinucleotide oxidase determines yogurt acidification rate.

Yogurt is made by fermenting milk with 2 lactic acid bacteria, Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus. To comprehensively understand the protocooperation mechanism between S. thermophilus and L. bulgaricus in yogurt fermentation, we examined 24 combinations of cocultures comprising 7 fast- or slow-acidifying S. thermophilus strains with 6 fast- or slow-acidifying L. bulgaricus strains. Furthermore, 3 NADH oxidase (Nox)-deficient mutants (Δnox) and one pyruvate formate-lyase deficient mutant (ΔpflB) of S. thermophilus were used to evaluate the factor that determines the acidification rate of S. thermophilus. The results revealed that the acidification rate of S. thermophilus monoculture determined the yogurt fermentation rates, despite the coexistence of L. bulgaricus, whose acidification rate was either fast or slow. Significant correlation was found between the acidification rate of S. thermophilus monoculture and the amount of formate production. Result using ΔpflB showed that the formate was indispensable for the acidification of S. thermophilus. Moreover, results of the Δnox experiments revealed that formate production required Nox activity, which not only regulated dissolved oxygen, but also the redox potential. The Nox provided the large decrease in redox potential required by pyruvate formate-lyase to produce formate. A highly significant correlation was found between formate accumulation and Nox activity in S. thermophilus. In conclusion, the formate production ability provided by the action of Nox activity determines the acidification rate of S. thermophilus, and consequently, regulates yogurt coculture fermentation.

A new method for producing superior set yogurt, focusing on heat treatment and homogenization.

Extended shelf life (ESL) processing (i.e., heat treatment at 130°C for 2 s) is usually not used for producing set yogurt because of the fragility of the curd structure. We investigated the effects of homogenization conducted at higher pressure than the conventional conditions (10 MPa for the first stage and 5 MPa for the second stage) on the curd structure of set yogurt, with a focus on the fat globule size. Each yogurt mix was adjusted at the range of fat globule sizes from 0.45 µm to 1.1 µm by a homogenizer and then heated at 95°C for 5 min (conventional heat treatment), 120°C for 2 s, ESL processing, or 140°C for 2 s. The yogurt mixes were fermented by a common yogurt starter, and the curd texture of the obtained yogurts was evaluated. We observed that the curd hardness and curd firmness of the yogurt were each negatively correlated with the fat globule size regardless of the heat-treatment temperature. Compared with the curd obtained with conventional heat treatment, the ESL-processed curd was extremely fragile, but significantly smooth. With ESL processing, a curd hardness >40 g, which is a sufficient strength for commercial transport systems, was obtained by making the fat particle size <0.6 µm, using 2-stage homogenization pressure: 35 MPa for the first stage and 5 MPa for the second stage. A microscopy analysis indicated that the smaller fat globules reinforce the network structure. The yogurt made by ESL processing and that created with 35 + 5 MPa homogenization had significant sensory evaluation scores. Our results indicate that the combination of ESL processing and 35 + 5 MPa homogenization is a novel and useful method for manufacturing set yogurt.

Ral GTPase Activation by Downregulation of RalGAP Enhances Oral Squamous Cell Carcinoma Progression.

Ral small GTPases, consisting of RalA and RalB, are members of the Ras family. Their activity is upregulated by RalGEFs. Since several RalGEFs are downstream effectors of Ras, Ral is activated by the oncogenic mutant Ras. Ral is negatively regulated by RalGAP complexes that consist of a catalytic α1 or α2 subunit and its common partner ß subunit and similarly regulate the activity of RalA as well as RalB in vitro. Ral plays an important role in the formation and progression of pancreatic and lung cancers. However, the involvement of Ral in oral squamous cell carcinoma (OSCC) is unclear. In this study, we investigated OSCC by focusing on Ral. OSCC cell lines with high Ral activation exhibited higher motility. We showed that knockdown of RalGAPß increased the activation level of RalA and promoted the migration and invasion of HSC-2 OSCC cells in vitro. In contrast, overexpression of wild-type RalGAPα2 in TSU OSCC cells attenuated the activation level of RalA and inhibited cell migration and invasion. Real-time quantitative polymerase chain reaction analysis of samples from patients with OSCC showed that RalGAPα2 was downregulated in oral cancer tissues as compared with normal epithelia. Among patients with OSCC, those with a lower expression of RalGAPα2 showed a worse overall survival rate. A comparison of DNA methylation and histone modifications of the RalGAPα2 gene in OSCC cell lines suggested that crosstalk among DNA methylation, histone H4Ac, and H3K27me2 was involved in the downregulation of RalGAPα2. Thus, activation of Ral GTPase by downregulation of RalGAP expression via a potential epigenetic mechanism may enhance OSCC progression.

The critical role of urease in yogurt fermentation with various combinations of Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus.

The protocooperation between Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus relies on metabolite exchanges that accelerate acidification during yogurt fermentation. Conflicting results have been obtained in terms of the effect of the Strep. thermophilus urease and the NH3 and CO2 that it generates on the rate of acidification in yogurt fermentation. It is difficult to perform a systematic study of the effects of urease on protocooperation because it is necessary to distinguish among the direct, indirect, and strain-specific effects resulting from the combination of the strains of both species. To evaluate the direct effects of urease on protocooperation, we generated 3 urease-deficient mutants (ΔureC) of fast- and slow-acidifying Strep. thermophilus strains and observed the effects of NH3 or CO2 supplementation on acidification by the ΔureC strains. Further, we examined 5 combinations of 3 urease-deficient ΔureC strains with 2 CO2-responsive or CO2-unresponsive strains of L. bulgaricus. Urease deficiency induced a shortage of ammonia nitrogen and CO2 for the fast- and slow-acidifying Strep. thermophilus and for the CO2-responsive L. bulgaricus, respectively. Notably, the shortage of ammonia nitrogen had more severe effects than that of CO2 on yogurt fermentation, even if coculture with L. bulgaricus masked the effect of urease deficiency. Our work established (1) that urease deficiency inhibits the fermentative acceleration of protocooperation regardless of the Strep. thermophilus and L. bulgaricus strain combinations, and (2) that urease is an essential factor for effective yogurt acidification.

One-dimensional α condensation of α-linear-chain states in ¹²C and ¹6O.

We present a new picture that the α-linear-chain structure for 12C and 16O has one-dimensional α condensate character. The wave functions of linear-chain states that are described by superposing a large number of Brink wave functions have extremely large overlaps of nearly 100% with single Tohsaki-Horiuchi-Schuck-Röpke wave functions, which were proposed to describe the α condensed "gaslike" states. Although this new picture is different from the conventional idea of the spatial localization of α clusters, the density distributions are shown to have localized α clusters due to the inter-α Pauli repulsion.

Oligomeric amyloid ß induces IL-1ß processing via production of ROS: implication in Alzheimer's disease.

Alzheimer's disease (AD) is a chronic neurodegenerative disease characterized by progressive neuronal loss and cognitive decline. Oligomeric amyloid ß (oAß) is involved in the pathogenesis of AD by affecting synaptic plasticity and inhibiting long-term potentiation. Although several lines of evidence suggests that microglia, the resident immune cells in the central nervous system (CNS), are neurotoxic in the development of AD, the mechanism whether or how oAß induces microglial neurotoxicity remains unknown. Here, we show that oAß promotes the processing of pro-interleukin (IL)-1ß into mature IL-1ß in microglia, which then enhances microglial neurotoxicity. The processing is induced by an increase in activity of caspase-1 and NOD-like receptor family, pyrin domain containing 3 (NLRP3) via mitochondrial reactive oxygen species (ROS) and partially via NADPH oxidase-induced ROS. The caspase-1 inhibitor Z-YVAD-FMK inhibits the processing of IL-1ß, and attenuates microglial neurotoxicity. Our results indicate that microglia can be activated by oAß to induce neuroinflammation through processing of IL-1ß, a pro-inflammatory cytokine, in AD.

Nonlocalized clustering: a new concept in nuclear cluster structure physics.

We investigate the α+^{16}O cluster structure in the inversion-doublet band (Kπ=0(1)±}) states of 20Ne with an angular-momentum-projected version of the Tohsaki-Horiuchi-Schuck-Röpke (THSR) wave function, which was successful "in its original form" for the description of, e.g., the famous Hoyle state. In contrast with the traditional view on clusters as localized objects, especially in inversion doublets, we find that these single THSR wave functions, which are based on the concept of nonlocalized clustering, can well describe the Kπ=0(1)- band and the Kπ=0(1)+ band. For instance, they have 99.98% and 99.87% squared overlaps for 1- and 3- states (99.29%, 98.79%, and 97.75% for 0+, 2+, and 4+ states), respectively, with the corresponding exact solution of the α+16O resonating group method. These astounding results shed a completely new light on the physics of low energy nuclear cluster states in nuclei: The clusters are nonlocalized and move around in the whole nuclear volume, only avoiding mutual overlap due to the Pauli blocking effect.

Downregulation of Ral GTPase-activating protein promotes tumor invasion and metastasis of bladder cancer.

The small GTPase Ral is known to be highly activated in several human cancers, such as bladder, colon and pancreas cancers. It is reported that activated Ral is involved in cell proliferation, migration and metastasis of bladder cancer. This protein is activated by Ral guanine nucleotide exchange factors (RalGEFs) and inactivated by Ral GTPase-activating proteins (RalGAPs), the latter of which consist of heterodimers containing a catalytic α1 or α2 subunit and a common ß subunit. In Ras-driven cancers, such as pancreas and colon cancers, constitutively active Ras mutant activates Ral through interaction with RalGEFs, which contain the Ras association domain. However, little is known with regard to the mechanism that governs aberrant activation of Ral in bladder cancer, in which Ras mutations are relatively infrequent. Here, we show that Ral was highly activated in invasive bladder cancer cells due to reduced expression of RalGAPα2, the dominant catalytic subunit in bladder, rather than increased expression of RalGEFs. Exogenous expression of wild-type RalGAPα2 in KU7 bladder cancer cells with invasive phenotype, but not mutant RalGAPα2-N1742K lacking RalGAP activity, resulted in attenuated cell migration in vitro and lung metastasis in vivo. Furthermore, genetic ablation of Ralgapa2 promoted tumor invasion in a chemically-induced murine bladder cancer model. Importantly, immunohistochemical analysis of human bladder cancer specimens revealed that lower expression of RalGAPα2 was associated with advanced clinical stage and poor survival of patients. Collectively, these results are highly indicative that attenuated expression of RalGAPα2 leads to disease progression of bladder cancer through enhancement of Ral activity.

Calculation of the surface potential and surface charge density by measurement of the three-phase contact angle.

The silica/silicon wafer is widely used in the semiconductor industry in the manufacture of electronic devices, so it is essential to understand its physical chemistry and determine the surface potential at the silica wafer/water interface. However, it is difficult to measure the surface potential of a silica/silicon wafer directly due to its high electric resistance. In the present study, the three-phase contact angle (TPCA) on silica is measured as a function of the pH. The surface potential and surface charge density at the silica/water surface are calculated by a model based on the Young-Lippmann equation in conjunction with the Gouy-Chapman model for the electric double layer. In measurements of the TPCA on silica, two distinct regions were identified with a boundary at pH 9.5-showing a dominance of the surface ionization of silanol groups below pH 9.5 and a dominance of the dissolution of silica into the aqueous solution above pH 9.5. Since the surface chemistry changes above pH 9.5, the model is applied to solutions below pH 9.5 (ionization dominant) for the calculation of the surface potential and surface charge density at the silica/aqueous interface. In order to evaluate the model, a galvanic mica cell was made of a mica sheet and the surface potential was measured directly at the mica/water interface. The model results are also validated by experimental data from the literature, as well as the results obtained by the potentiometric titration method and the electro-kinetic measurements.

Short communication: effect of oxygen on symbiosis between Lactobacillus bulgaricus and Streptococcus thermophilus.

Lactobacillus delbrueckii ssp. bulgaricus (L. bulgaricus) and Streptococcus thermophilus are traditionally used for the manufacture of yogurt. It is said that a symbiotic relationship exists between Strep. thermophilus and L. bulgaricus and this decreases fermentation time. It is well known that L. bulgaricus is stimulated by the formate produced by Strep. thermophilus, and Strep. thermophilus is stimulated by free amino acids and peptides liberated from milk proteins by L. bulgaricus in symbiotic fermentation. We found that acid production by starter culture LB81 composed of L. bulgaricus 2038 and Strep. thermophilus 1131 was greatly accelerated by decreasing dissolved oxygen (DO) to almost 0 mg/kg in the yogurt mix (reduced dissolved oxygen fermentation) and that DO interferes with the symbiotic relationship between L. bulgaricus 2038 and Strep. thermophilus 1131. We attributed the acceleration of acid production of LB81 by reduced dissolved oxygen fermentation mainly to the acceleration of formate production and the suppression of acid production of LB81 by DO mainly to the suppression of formate production.

IgG4-related inflammatory pseudotumor of the trigeminal nerve: another component of IgG4-related sclerosing disease?

IgG4-related IPTs have been reported in various sites and may form part of the spectrum of systemic IgG4-related sclerosing disease. Some pseudotumors are clinically and radiologically indistinguishable from malignant tumors. We present the first case of an IgG4-related IPT of the trigeminal nerve diagnosed histopathologically without involvement of any of the common sites. The trigeminal nerve pseudotumor may represent a component of IgG4-related sclerosing disease.

Adenosine A1 receptor agonists reduce hyperalgesia after spinal cord injury in rats.

STUDY DESIGN: An in vivo study using a spinal cord compression model in rats. OBJECTIVES: To evaluate the effect of adenosine on thermal hyperalgesia after spinal cord injury (SCI). SUMMARY OF BACKGROUND DATA: After SCI, some patients suffer dysesthesia that is unresponsive to conventional treatments. We previously established a rat thoracic spinal cord mild-compression model by which we were able to induce thermal hyperalgesia in the hind limbs. METHODS: The thoracic spinal cord was compressed gently using a 20-g weight for 20 min. The withdrawal latency in response to thermal stimulation was monitored bilaterally in the hind limbs using Hargreaves' Plantar test apparatus. RESULTS: SCI-induced thermal hyperalgesia was mimicked by the intrathecal application of 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), a selective adenosine A1 receptor antagonist. Hyperalgesia induced by SCI was significantly inhibited by the intrathecal application of 10-30 nmol chloro-adenosine (Cl-adenosine), a nonselective adenosine receptor agonist. The effect of Cl-adenosine (10 nmol) on hyperalgesia after SCI was blocked by the simultaneous application of DPCPX. Intrathecal application of R(-)N6-(2phenylisopropyl) adenosine (R-PIA; 10 nmol), a selective A1 receptor agonist, also inhibited SCI-induced hyperalgesia. In contrast, intrathecal application of CGS21680, a selective adenosine A2a receptor agonist, did not inhibit SCI-induced hyperalgesia. CONCLUSIONS: These results suggest that adenosine inhibits hyperalgesia through the stimulation of A1 receptors. Adenosine or adenosine A1 receptor agonists should be considered as candidates for new therapeutic methods for treating post-SCI dysesthesia.

A method for manufacturing superior set yogurt under reduced oxygen conditions.

The yogurt starters Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus are well-known facultatively anaerobic bacteria that can grow in oxygenated environments. We found that they removed dissolved oxygen (DO) in a yogurt mix as the fermentation progressed and that they began to produce acid actively after the DO concentration in the yogurt mix was reduced to 0 mg/kg, suggesting that the DO retarded the production of acid. Yogurt fermentation was carried out at 43 or 37 degrees C both after the DO reduction treatment and without prior treatment. Nitrogen gas was mixed and dispersed into the yogurt mix after inoculation with yogurt starter culture to reduce the DO concentration in the yogurt mix. The treatment that reduced DO concentration in the yogurt mix to approximately 0 mg/kg beforehand caused the starter culture LB81 used in this study to enter into the exponential growth phase earlier. Furthermore, the combination of reduced DO concentration in the yogurt mix beforehand and incubation at a lower temperature (37 degrees C) resulted in a superior set yogurt with a smooth texture and strong curd structure.

alpha-particle condensation in 16O studied with a full four-body orthogonality condition model calculation.

To explore the four-alpha-particle condensate state in 16O, we solve a full four-body equation of motion based on the four-alpha-particle orthogonality condition model in a large four-alpha-particle model space spanned by Gaussian basis functions. A full spectrum up to the 0_{6};{+} state is reproduced consistently with the lowest six 0;{+} states of the experimental spectrum. The 0_{6};{+} state is obtained at about 2 MeV above the four-alpha-particle breakup threshold and has a dilute density structure, with a radius of about 5 fm. The state has an appreciably large alpha condensate fraction of 61%, and a large component of alpha+12C(0_{2};{+}) configuration, both features being reliable evidence for this state to be of four-alpha-particle condensate nature.

The long-term outcome of high tibial osteotomy: a ten- to 20-year follow-up.

We carried out a prospective study of 132 patients (159 knees) who underwent closed-wedge high tibial osteotomy for severe medial compartment osteoarthritis between 1988 and 1997. A total of 94 patients (118 knees) was available for review at a mean of 16.4 years (16 to 20). Seven patients (7.4%) (11 knees) required conversion to total knee replacement. Kaplan-Meier survival was 97.6% (95% confidence interval 95.0 to 100) at ten years and 90.4% (95% confidence interval 84.1 to 96.7) at 15 years. Excellent and good results as assessed by the Hospital for Special Surgery knee score were achieved in 87 knees (73.7%). A pre-operative body mass index > 27.5 kg/m(2) and range of movement < 100 degrees were risk factors predicting early failure. Although our long-term results were satisfactory, strict indications for osteotomy are required if long-term survival is required.

Immunomodulatory effects of polysaccharides produced by Lactobacillus delbrueckii ssp. bulgaricus OLL1073R-1.

The extracellular polysaccharides (EPS) produced by lactic acid bacteria (LAB) are associated with the rheology, texture, and mouthfeel of fermented milk products, including yogurt. This study investigated the immunomodulatory effects of EPS purified from the culture supernatant of Lactobacillus delbrueckii ssp. bulgaricus (L. bulgaricus) OLL1073R-1. The crude EPS were prepared from the culture supernatant of L. bulgaricus OLL1073R-1 by standard chromatographic methods, and were fractionated into neutral EPS and acidic EPS (APS). Acidic EPS were further fractionated into high molecular weight APS (H-APS) and low molecular weight APS (L-APS). High molecular weight APS were shown to be phosphopolysaccharides containing D-glucose, D-galactose, and phosphorus. Stimulation of mouse splenocytes by H-APS significantly increased interferon-gamma production, and, moreover, orally administered H-APS augmented natural killer cell activity. Oral administration of yogurt fermented with L. bulgaricus OLL1073R-1 and Streptococcus thermophilus OLS3059 to mice showed a similar level of immunomodulation as H-APS. However, these effects were not detected following administration of yogurt fermented with the starter combination of L. bulgaricus OLL1256 and S. thermophilus OLS3295. We conclude from these findings that yogurt fermented with L. bulgaricus OLL1073R-1, containing immunostimulative EPS, would have an immunomodulatory effect on the human body.

The wide distribution of endornaviruses, large double-stranded RNA replicons with plasmid-like properties.

The International Committee on Taxonomy of Viruses (ICTV) recently accepted Endornavirus as a new genus of plant dsRNA virus. We have determined the partial nucleotide sequences of the RNA-dependent RNA polymerase regions from the large dsRNAs (about 14 kbp) isolated from barley (Hordeum vulgare), kidney bean (Phaseolus vulgaris), melon (Cucumis melo), bottle gourd (Lagenaria siceraria), Malabar spinach (Basella alba), seagrass (Zostera marina), and the fungus Helicobasidium mompa. Phylogenetic analyses of these seven dsRNAs indicate that these dsRNAs are new members of the genus Endornavirus that are widely distributed over the plant and fungal kingdoms.

Genomic sequence of the pathogenic and allergenic filamentous fungus Aspergillus fumigatus.

Aspergillus fumigatus is exceptional among microorganisms in being both a primary and opportunistic pathogen as well as a major allergen. Its conidia production is prolific, and so human respiratory tract exposure is almost constant. A. fumigatus is isolated from human habitats and vegetable compost heaps. In immunocompromised individuals, the incidence of invasive infection can be as high as 50% and the mortality rate is often about 50% (ref. 2). The interaction of A. fumigatus and other airborne fungi with the immune system is increasingly linked to severe asthma and sinusitis. Although the burden of invasive disease caused by A. fumigatus is substantial, the basic biology of the organism is mostly obscure. Here we show the complete 29.4-megabase genome sequence of the clinical isolate Af293, which consists of eight chromosomes containing 9,926 predicted genes. Microarray analysis revealed temperature-dependent expression of distinct sets of genes, as well as 700 A. fumigatus genes not present or significantly diverged in the closely related sexual species Neosartorya fischeri, many of which may have roles in the pathogenicity phenotype. The Af293 genome sequence provides an unparalleled resource for the future understanding of this remarkable fungus.

Polymerase chain reaction-based analysis using deaminated DNA of dodecamer expansions in CSTB, associated with Unverricht-Lundborg myoclonus epilepsy.

Progressive myoclonus epilepsy of the Unverricht-Lundborg type is an autosomal recessive disorder that is characterized clinically by myoclonic seizures and ataxia. The majority of affected individuals carry repeat expansions of a dodecamer in the promoter region of the cystatin B gene. The unusually high GC content of this tract is refractory to conventional polymerase chain reaction (PCR), and, as a result, a circumventive procedure involving the deamination of DNA with sodium bisulfite has been proposed. This study evaluates the effectiveness of this deamination modification for the detection of dodecamer repeat variants. An analysis of 258 healthy Japanese individuals revealed an allele with four copies of the dodecamer repeat with a frequency of 0.01, in addition to the more commonly observed two and three copy repeat alleles. Homozygous repeat expansions 600 and 680 base pairs in length were detected in the analyses of two affected individuals. For these cases, sequencing, along with an alternative PCR-stutter formation, revealed 41 and 48 copies, respectively, of the dodecamer repeat. The complete conversion of C to T was observed in the expanded tracts, indicating that no methylation occurred at the CpG sites. Based on these results, it was concluded that the use of deaminated DNA allows for a precise analysis of consecutive GC tracts.

Synthetic biodegradable polymers as drug delivery systems for bone morphogenetic proteins.

Bone morphogenetic proteins (BMP) induce bone formation in vivo, and clinical application in repair of bone fractures and defects is expected. However, appropriate systems to deliver BMP for clinical use need to be developed. We synthesized a new synthetic biodegradable polymer, poly-D,L-lactic acid-para-dioxanone-polyethylene glycol block copolymer (PLA-DX-PEG), to serve as a biocompatible, biodegradable polymer for recombinant human (rh) BMP-2 delivery systems. In animal experiments, new bone was efficiently formed and a large bone defect was repaired using PLA-DX-PEG/rhBMP-2 composites. In addition, this new polymer could be used as an injectable delivery system for rhBMP-2. The rhBMP-2/PLA-DX-PEG composites also could be combined with other materials such as hydroxyapatite or titanium. This new synthetic polymer might be used for rhBMP-2 delivery in various clinical situations involving repair of bone, leading to great changes in orthopedic treatment.