Limited Tryptic Degradation of Urea Denatured Glycinin

Digestibilities of native, 5 m urea-denatured and 8 m urea-denatured glycinin were studied. Urea was removed by dialysis before digestion. The tryptic digestion of the proteins are influenced by ionic strength. Under low ionic strength condition (0 m NaCl), the proteins, even native glycinin, are well degraded. On the other hand, under high ionic strength condition (0.5 m NaCl), native glycinin resists the tryptic attack and 5 m urea-denatured glycinin is best degraded. The digestibility of 8 m urea-denatured glycinin is lower than that of 5 m urea-denatured one under the condition. The gel filtration and electrophoretic properties show that the digestion intermediate like glycinin-T (the intermediate from native glycinin) is contained in the digestion products. These suggest that the urea-denatured protein contains the almost renatured component after removal of urea. A larger amount of the glycinin-T-like protein was detected at 8 m urea denaturation than at 5 m urea. Therefore, glycinin renatures more readily from 8 m urea denaturation. Probably this is the cause of the decreased digestibility at 8 m urea denaturation.     

On Basic Subunits Dissociated from C (11S) Component of Soybean Proteins with Urea

Galactolipase (galactolipid acyl hydrolase, EC 3.1.1.26) was purified 147-fold in good yield (91 %) from rice bran by affinity chromatography, in which the enzyme was adsorbed on a palmitoylated gauze column at pH 5.5 and then was eluted with a buffer solution containing a detergent such as sodium deoxycholate or Triton X–100 at pH 8.0. The preparation obtained was further purified by gel filtration on a Sephadex G–100 column and isoelectric focusing. After electrophoresis, the enzyme separated into four components with different isoelectric points. It seems that galactolipase in rice bran exists in multiple forms. The major component (G–2) with isoelectric point of 7.3, one of them, was purified 268-fold and electrophoretically homogeneous. The enzyme (G–2) hydrolyzed rapidly galactolipid and also slowly phospholipid, but hardly triglyceride.     

Marginal iron deficiency enhances liver triglyceride accumulation in rats fed a high-sucrose diet

ABSTRACT

We investigated whether marginal iron-deficiency (MID) without anemia influences liver lipid accumulation in rats. Ingestion of a MID diet in which the iron concentration was half of AIN-93 formulation (iron-adequate, IA) for 3 weeks decreased liver iron concentration without anemia. We then evaluated the influence of the MID diet on liver lipid accumulation in combination with a high-sucrose (HS) diet and confirmed that the HS-MID diet successfully decreased liver iron concentration without anemia. Additionally, a significant increase in liver triglyceride concentration was found, accompanied by upregulation of hepatic fatty acid synthase expression in the rats fed the HS-MID diet compared to those in the rats fed an HS-IA diet, although no difference was observed in plasma transaminase activity and hepatic interleukin-1β expression. These results suggest that MID enhances de novo lipid synthesis via upregulation of lipogenic gene expression in combination with sucrose in the diet.

Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; HS, high sucrose; IA, iron adequate; ID, iron deficiency; MID, marginal irondeficiency; NAFLD, non-alcoholic fatty liver disease     

Synergistic co-activation in multi-directional postural control in humans

To examine the muscle synergies of multi-directional postural control, we calculated the target-directed variance fraction (η) and net action direction of each muscle using the electromyogram-weighted averaging (EWA) method. Subjects stood barefoot on a force platform and maintained their posture by producing a center of pressure (COP) in twelve target directions. Surface electromyograms were recorded from 6 right-sided muscles: tibialis anterior (TA), soleus (SOL), lateral gastrocnemius (LG), medial gastrocnemius (MG), fibularis longus (FL), and gluteus medius (GM). η was calculated from COP with duration of 20-s, during which the COP was relatively constant. The EWA method was applied to the EMG and the two COP components to estimate the net action direction of each muscle. The results showed that η values in all directions did not cross the 0.8 threshold. This suggests that human postural control is achieved by synergistic co-activation. The EWA revealed that the net action directions of TA, SOL, LG, MG, and GM were 277.6°, 71.1°, 87.7°, 94.0°, and 2.2°, respectively. This suggests that postural maintenance by muscle synergy can be attributed to the relevant muscles having various action directions. These results demonstrate that muscle synergies can be investigated using COP fluctuations.     

Development of a Highly Sensitive Immuno-PCR Assay for the Measurement of α-Galactosidase A Protein Levels in Serum and Plasma

Fabry disease is an X-linked genetic disorder caused by defects in the α-galactosidase A (GLA) gene, and heterogeneous mutations lead to quantitative and/or qualitative defects in GLA protein in male patients with Fabry disease. Random X-chromosomal inactivation modifies the clinical and biochemical features of female patients with Fabry disease. Functional polymorphisms have been frequently reported in recent times, and these increase the difficulty of understanding the pathogenetic basis of the disease. To date, GLA protein level has been measured using an enzyme-linked immunosorbent assay (ELISA). However, ELISA is not highly sensitive due to the high background noise. In this paper, we introduce a novel application of the immuno-polymerase chain reaction (PCR) method (termed Multiple Simultaneous Tag [MUSTag]) for measurement of the GLA protein level in blood samples. We compared the sensitivities of the MUSTag method with plates or magnetic beads with those of ELISA for recombinant human GLA and found that the apparent maximal sensitivity was higher for the former than for the latter. We then measured the GLA concentrations in serum and plasma from male patients with classic Fabry disease (Male Fabry), females with Fabry disease (Female Fabry), male subjects harboring the functional polymorphism p.E66Q (E66Q), and control (Control) subjects. Our results revealed that compared to the MUSTag plate and ELISA, the MUSTag beads assay afforded a clearer estimation of the GLA protein levels in the serum and plasma with minimal or no background noise, although all the methods could differentiate between the Male Fabry, E66Q, and Control groups. The Female Fabry group showed characteristic heterogeneity, which was consistent with the X-linked inheritance. This novel method is expected to be useful for the sensitive determination of GLA level in blood and elucidation of the pathogenetic basis of Fabry disease.     

Expansion of Activated Memory CD4+ T Cells Affects Infectivity of CCR5-Tropic HIV-1 in Humanized NOD/SCID/JAK3null Mice

Humanized mice reconstituted with human hematopoietic cells have been developed as an experimental animal model for human immunodeficiency virus type 1 (HIV-1) infection. Myeloablative irradiation is usually performed to augment the engraftment of donor hematopoietic stem cells (HSCs) in recipient mice; however, some mouse strains are susceptible to irradiation, making longitudinal analysis difficult. We previously attempted to construct humanized NOD/SCID/JAK3^null (hNOJ) mice, which were not irradiated prior to human HSC transplantation. We found that, over time, many of the reconstituted CD4⁺ T cells expanded with an activated effector memory phenotype. Therefore, the present study used hNOJ mice that were irradiated (hNOJ (IR+)) or not (hNOJ (IR−)) prior to human HSC transplantation to examine whether the development and cellularity of the reconstituted CD4⁺ T cells were influenced by the degree of chimerism, and whether they affected HIV-1 infectivity. Indeed, hNOJ (IR+) mice showed a greater degree of chimerism than hNOJ (IR−) mice. However, the conversion of CD4⁺ T cells to an activated effector memory phenotype, with a high percentage of cells showing Ki-67 expression, occurred in both hNOJ (IR+) and hNOJ (IR−) mice, probably as a result of lymphopenia-induced homeostatic expansion. Furthermore, when hNOJ (IR+) and hNOJ (IR−) mice, which were selected as naïve- and memory CD4⁺ T cell subset-rich groups, respectively, were infected with CCR5-tropic HIV-1 in vivo, virus replication (as assessed by the plasma viral load) was delayed; however, the titer subsequently reached a 1-log higher level in memory-rich hNOJ (IR−) mice than in naïve-rich hNOJ (IR+) mice, indicating that virus infectivity in hNOJ mice was affected by the different status of the reconstituted CD4⁺ T cells. Therefore, the hNOJ mouse model should be used selectively, i.e., according to the specific experimental objectives, to gain an appropriate understanding of HIV-1 infection/pathogenesis.     

Biphasic regulation of levofloxacin on lipopolysaccharide-induced IL-1beta production

Fluoroquinolones have been known to exert modulatory activity on immune responses to microbial infection. However, the mechanism of this immunomodulation has not been well elucidated. In this study, we investigated the effect of levofloxacin on lipopolysaccharide (LPS)-induced production of interleukin-1beta (IL-1beta) in RAW264.7 cells. We showed that LPS-stimulated release of pre-synthesized IL-1beta was promoted by levofloxacin, in part via the p38 mitogen-activated protein kinase (MAPK) pathway. On the other hand, newly synthesized IL-1beta production was inhibited by levofloxacin. This immunoregulatory function of levofloxacin in the later phase as well as promotion of pre-synthesized IL-1beta release by levofloxacin in the early phase might be advantageous in the host defense to microbial pathogens.     

Embryo spacing and implantation timing are differentially regulated by LPA3-mediated lysophosphatidic acid signaling in mice

In polytocous animals, blastocysts are evenly distributed along each uterine horn and implant. The molecular mechanisms underlying these precise events remain elusive. We recently showed that lysophosphatidic acid (LPA) has critical roles in the establishment of early pregnancy by affecting embryo spacing and subsequent implantation through its receptor, LPA3. Targeted deletion of Lpa3 in mice resulted in delayed implantation and embryo crowding, which is associated with a dramatic decrease in the prostaglandins and prostaglandin-endoperoxide synthase 2 expression levels. Exogenous administration of prostaglandins rescued the delayed implantation but did not rescue the defects in embryo spacing, suggesting the role of prostaglandins in implantation downstream of LPA3 signaling. In the present study, to know how LPA3 signaling regulates the embryo spacing, we determined the time course distribution of blastocysts during the preimplantation period. In wild-type (WT) uteri, blastocysts were distributed evenly along the uterine horns at Embryonic Day 3.8 (E3.8), whereas in the Lpa3-deficient uteri, they were clustered in the vicinity of the cervix, suggesting that the mislocalization and resulting crowding of the embryos are the cause of the delayed implantation. However, embryos transferred singly into E2.5 pseudopregnant Lpa3-deficient uterine horns still showed delayed implantation but on-time implantation in WT uteri, indicating that embryo spacing and implantation timing are two segregated events. We also found that an LPA3-specific agonist induced rapid uterine contraction in WT mice but not in Lpa3-deficient mice. Because the uterine contraction is critical for embryo spacing, our results suggest that LPA3 signaling controls embryo spacing via uterine contraction around E3.5.     

Performance comparison of second- and third-generation sequencers using a bacterial genome with two chromosomes

Background

The availability of diverse second- and third-generation sequencing technologies enables the rapid determination of the sequences of bacterial genomes. However, identifying the sequencing technology most suitable for producing a finished genome with multiple chromosomes remains a challenge. We evaluated the abilities of the following three second-generation sequencers: Roche 454 GS Junior (GS Jr), Life Technologies Ion PGM (Ion PGM), and Illumina MiSeq (MiSeq) and a third-generation sequencer, the Pacific Biosciences RS sequencer (PacBio), by sequencing and assembling the genome of Vibrio parahaemolyticus, which consists of a 5-Mb genome comprising two circular chromosomes.

Results

We sequenced the genome of V. parahaemolyticus with GS Jr, Ion PGM, MiSeq, and PacBio and performed de novo assembly with several genome assemblers. Although GS Jr generated the longest mean read length of 418 bp among the second-generation sequencers, the maximum contig length of the best assembly from GS Jr was 165 kbp, and the number of contigs was 309. Single runs of Ion PGM and MiSeq produced data of considerably greater sequencing coverage, 279× and 1,927×, respectively. The optimized result for Ion PGM contained 61 contigs assembled from reads of 77× coverage, and the longest contig was 895 kbp in size. Those for MiSeq were 34 contigs, 58× coverage, and 733 kbp, respectively. These results suggest that higher coverage depth is unnecessary for a better assembly result. We observed that multiple rRNA coding regions were fragmented in the assemblies from the second-generation sequencers, whereas PacBio generated two exceptionally long contigs of 3,288,561 and 1,875,537 bps, each of which was from a single chromosome, with 73× coverage and mean read length 3,119 bp, allowing us to determine the absolute positions of all rRNA operons.

Conclusions

PacBio outperformed the other sequencers in terms of the length of contigs and reconstructed the greatest portion of the genome, achieving a genome assembly of “finished grade” because of its long reads. It showed the potential to assemble more complex genomes with multiple chromosomes containing more repetitive sequences.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-699) contains supplementary material, which is available to authorized users.