Highly efficient protein expression of Plasmodium vivax surface antigen, Pvs25, by silkworm and its biochemical analysis.

Plasmodium vivax ookinete surface protein, Pvs25, is a candidate for a transmission-blocking vaccine (TBV) for malaria. Pvs25 has four EGF-like domains containing 22 cysteine residues forming 11 intramolecular disulfide bonds, a structural feature that makes its recombinant protein expression difficult. In this study, we report the high expression of recombinant Pvs25 as a soluble form in silkworm, Bombyx mori. The Pvs25 protein was purified from hemolymphs of larvae and pupae by affinity chromatography. In the Pvs25 expressed by silkworm, no isoforms with inappropriate disulfide bonds were found, requiring no further purification step, which is necessary in the case of Pichia pastoris-based expression systems. The Pvs25 from silkworm was confirmed to be molecularly uniform by sodium dodecyl sulfate gel electrophoresis and size-exclusion chromatography. To examine the immunogenicity, the Pvs25 from B. mori was administered to BALB/c mice subcutaneously with oil adjuvant. The Pvs25 produced by silkworm induced potent and robust immune responses, and the induced antisera correctly recognized P. vivax ookinetes in vitro, demonstrating the potency of Pvs25 from silkworm as a candidate for a malaria TBV. To the best of our knowledge, this is the first study to construct a system for mass-producing malaria TBV antigens using silkworm.

Morning Bright Light Treatment for Chronic Low Back Pain: Potential Impact on the Volatility of Pain, Mood, Function, and Sleep.

OBJECTIVES: Most treatment outcome studies for people with chronic low back pain (CLBP) have based analyses on and reported only the mean levels of these factors. However, high levels of pain, mood, function, and sleep volatility may represent unique factors contributing to diminished quality of life. Our goal was to determine whether bright light treatment affected both mean levels of pain, mood, function, and sleep and reduced volatility in these outcomes. METHODS: US military veterans with CLBP (N = 22) underwent an open trial with a seven-day baseline, followed by 13 days of a one-hour morning bright light treatment self-administered at their home and a 30-day follow-up. Participants completed daily diary measures at 12 Pm and 6 Pm every day during the three study epochs. RESULTS: Using location scale modeling, results suggested that, in addition to being associated with changes in mean levels of pain intensity, pain interference, negative affect, and sleep quality, bright light treatment was also related to reductions in the volatility of pain intensity and negative affect, reductions that were largely maintained during follow-up. CONCLUSIONS: Changes in mean levels and volatility were independent factors, suggesting that bright light treatment was related to participants experiencing fewer "pain flares." These findings underscore the potential importance of volatility as a future treatment target.

An Open Trial of Morning Bright Light Treatment Among US Military Veterans with Chronic Low Back Pain: A Pilot Study.

OBJECTIVE: To examine the feasibility, acceptability, and effects of a home-based morning bright light treatment on pain, mood, sleep, and circadian timing in US veterans with chronic low back pain. DESIGN: An open treatment trial with a seven-day baseline, followed by 13 days of a one-hour morning bright light treatment self-administered at home. Pain, pain sensitivity, mood, sleep, and circadian timing were assessed before, during, and after treatment. SETTING: Participants slept at home, with weekly study visits and home saliva collections. PARTICIPANTS: Thirty-seven US veterans with medically verified chronic low back pain. METHODS: Pain, mood, and sleep quality were assessed with questionnaires. Pain sensitivity was assessed using two laboratory tasks: a heat stimulus and an ischemia stimulus that gave measures of threshold and tolerance. Sleep was objectively assessed with wrist actigraphy. Circadian timing was assessed with the dim light melatonin onset. RESULTS: Morning bright light treatment led to reduced pain intensity, pain behavior, thermal pain threshold sensitivity, post-traumatic stress disorder symptoms, and improved sleep quality (P < 0.05). Phase advances in circadian timing were associated with reductions in pain interference (r = 0.55, P < 0.05). CONCLUSIONS: Morning bright light treatment is a feasible and acceptable treatment for US veterans with chronic low back pain. Those who undergo morning bright light treatment may show improvements in pain, pain sensitivity, and sleep. Advances in circadian timing may be one mechanism by which morning bright light reduces pain. Morning bright light treatment should be further explored as an innovative treatment for chronic pain conditions.

Do the Morningness-Eveningness Questionnaire and Munich ChronoType Questionnaire Change After Morning Light Treatment?

The Morningness-Eveningness Questionnaire (MEQ) and Munich ChronoType Questionnaire (MCTQ) are sometimes used to estimate circadian timing. However, it remains unclear if they can reflect a change in circadian timing after a light treatment. In this study, 31 participants (25-68 years) completed both questionnaires before and after a 13-28 day morning light treatment. The dim light melatonin onset (DLMO), a physiological marker of circadian timing, was also assessed in a subsample of 16 participants. The DLMO phase advanced on average by 47 minutes (p<0.001). The MEQ score increased by 1.8 points (p=0.046). The MSFsc measure derived from the MCTQ advanced by 8.7 minutes (p=0.17). The shift towards morningness observed in both questionnaires correlated with the phase advance observed in the DLMO (MEQ r=-0.46, p=0.036; MSFsc r=0.81, p<0.001). Results suggest that these circadian questionnaires can change in response to a light treatment, indicating they can reflect underlying changes in circadian timing.

Detoxification of selenite to form selenocyanate in mammalian cells.

When human hepatoma HepG2 cells were exposed to sodium selenite, an unknown selenium metabolite was detected in the cytosolic fraction by HPLC-inductively coupled plasma mass spectrometry (ICP-MS). The unknown selenium metabolite was also detected in the mixture of HepG2 homogenate and sodium selenite in the presence of exogenous glutathione (GSH). The unknown selenium metabolite was identified as selenocyanate by electrospray ionization mass spectrometry (ESI-MS) and ESI quadrupole time-of-flight mass spectrometry (ESI-Q-TOF-MS). Because exogenous cyanide increased the amount of selenocyanate in the mixture, selenocyanate seemed to be formed by the reaction between selenide or its equivalent, the product of the reduction of selenite, and endogenous cyanide. Rhodanase, an enzyme involved in thiocyanate synthesis, was not required for the formation of selenocyanate. Selenocyanate was less toxic to HepG2 cells than selenite or cyanide, suggesting that it was formed to reduce the toxicity of selenite. However, selenocyanate could be assimilated into selenoproteins and selenometabolites in rats in the same manner as selenite. Consequently, selenite was metabolized to selenocyanate to temporarily ameliorate its toxicity, and selenocyanate acted as an intrinsic selenium pool in cultured cells exposed to surplus selenite.

NF-E2 domination over Nrf2 promotes ROS accumulation and megakaryocytic maturation.

In megakaryocytes, the maturation process and oxidative stress response appear to be closely related. It has been suggested that increased oxygen tension and reactive oxygen species (ROS) promote megakaryopoiesis and that the expression of stress-responsive genes responsible for ROS elimination declines during megakaryocytic maturation. NF-E2 p45 is an essential regulator of megakaryopoiesis, whereas Nrf2 is a key activator of stress-responsive genes. Because p45 and Nrf2 have similar DNA-binding specificities, we hypothesized that p45 competes with Nrf2 to repress stress-responsive genes and achieves favorable intracellular conditions to allow ROS to be efficiently used as signaling molecules. We conducted comprehensive gene expression profiling with wild-type and p45-null megakaryocytes and examined the functional relationship between p45 and Nrf2. We found that 2 characteristic gene clusters are defined within p45 target genes: platelet genes and cytoprotective genes. The former are unique targets activated by p45, whereas the latter are common targets of p45 and Nrf2. Further analysis suggested that, as a less efficacious activator, p45 maintains moderate expression of cytoprotective genes through competing with Nrf2 and promotes ROS accumulation. Increased ROS enhanced platelet gene expression. These results suggest that p45 dominates over Nrf2 to enhance megakaryocytic maturation by promoting ROS accumulation.

Structural basis of alternative DNA recognition by Maf transcription factors.

Maf transcription factors constitute a family of the basic region-leucine zipper (bZip) factors and recognize unusually long DNA motifs (13 or 14 bp), termed the Maf recognition element (MARE). The MARE harbors extended GC sequences on each side of its core motif, which is similar to TRE or CRE (7 or 8 bp) recognized by the AP1 and CREB/ATF families, respectively. To ascertain the structural basis governing the acquirement of such unique DNA recognition, we determined the crystal structure of the MafG-DNA complex. Each MafG monomer consists of three helices in which the carboxyl-terminal long helix organizes one DNA-contacting element and one coiled-coil dimer formation element. To our surprise, two well-conserved residues, Arg57 and Asn61 in the basic region, play critical roles in Maf-specific DNA recognition. These two residues show unique side-chain orientations and interact directly with the extended GC bases. Maf-specific residues in the amino-terminal and basic regions appear to indirectly stabilize MARE recognition through DNA backbone phosphate interactions. This study revealed an alternative DNA recognition mechanism of the bZip factors that bestows specific target gene profiles upon Maf homodimers or Maf-containing heterodimers.

Reproducibility of maxillofacial anatomic landmarks on 3-dimensional computed tomographic images determined with the 95% confidence ellipse method.

OBJECTIVE: To assess the plotting reproducibility of landmarks on 3-dimensional computed tomography (3D-CT) images through use of the 95% confidence ellipse in order to propose sufficiently stable coordinate systems for 3D-CT measurements. MATERIALS AND METHODS: Six dentists plotted 19 landmarks twice on 3D-CT images. Scatterplots and the 95% ellipses were produced 3-dimensionally, and the areas of the ellipses were calculated for evaluating the reproducibility of landmarks. RESULTS: The plotting reproducibility of each landmark showed characteristic features. Among five landmarks (the sella [S], nasion [N], basion [Ba], orbitale [Or], and true porion [Po]) that are frequently used as reference points on cephalograms, Ba showed the smallest areas for all three coordinate axes, indicating high reproducibility. The coronoid process (CP) and the tooth-related landmarks showed relatively high reproducibility. CONCLUSION: Sufficiently stable coordinate axes could be proposed for different treatments and studies.

Molecular basis distinguishing the DNA binding profile of Nrf2-Maf heterodimer from that of Maf homodimer.

Nrf2-small Maf heterodimer activates the transcription of many cytoprotective genes through the antioxidant response element and serves as a key factor in xenobiotic and oxidative stress responses. Our surface plasmon resonance-microarray binding analysis revealed that both Nrf2-MafG heterodimer and MafG homodimer bind to the consensus Maf recognition element with high affinity but bind differentially to the suboptimal binding sequences degenerated from the consensus. We examined the molecular basis distinguishing the binding profile of Nrf2-MafG heterodimer from that of MafG homodimer and found that the Ala-502 residue in the basic region of Nrf2 is a critical determinant of its binding specificity. In Maf proteins, a tyrosine resides in the position corresponding to Ala-502 in Nrf2. We prepared a mutant Nrf2 molecule in which Ala-502 was replaced with tyrosine. In surface plasmon resonance-microarray analysis, heterodimer of Nrf2(A502Y) and MafG displayed a binding specificity similar to that of MafG homodimer. The target genes activated by mutant Nrf2(A502Y)-small Maf heterodimer were largely different, albeit with some overlap, from those activated by wild-type Nrf2-small Maf, indicating that the array of target genes regulated by Nrf2-small Maf heterodimer differs substantially from that regulated by Maf homodimer in vivo. These results suggest that the distinct DNA binding profile of Nrf2-Maf heterodimer is biologically significant for Nrf2 to function as a key regulator of cytoprotective genes. Our contention is supported that the differential DNA binding specificity between Maf homodimers and Nrf2-Maf heterodimers establishes the differential gene regulation by these dimer-forming transcription factors.

Phytoestrogens genistein and daidzin enhance the acetylcholinesterase activity of the rat pheochromocytoma cell line PC12 by binding to the estrogen receptor.

Some compounds derived from plants have been known to possess estrogenic properties and can thus alter the physiology of higher organisms. Genistein and daidzin are examples of these phytoestrogens, which have recently been the subject of extensive research. In this study, genistein and daidzin were found to enhance the acetylcholinesterase (AChE) activity of the rat neuronal cell line PC12 at concentrations as low as 0.08 muM by binding to the estrogen receptor (ER). Results have shown that this enhancement was effectively blocked by the known estrogen receptor antagonist tamoxifen, indicating the involvement of the ER in AChE induction. That genistein and daidzin are estrogenic were confirmed in a cell proliferation assay using the human breast cancer cell line MCF7. This proliferation was also blocked by tamoxifen, again indicating the involvement of the ER. On the other hand, incubating the PC12 cells in increasing concentrations of 17 beta-estradiol (E2) did not lead to enhanced AChE activity, even in the presence of genistein or daidzin. This suggests that mere binding of an estrogenic compound to the ER does not necessarily lead to enhanced AChE activity. Moreover, the effect of the phytoestrogens on AChE activity cannot be expressed in the presence of E2 since they either could not compete with the natural ligand in binding to the ER or that E2 down-regulates its own receptor. This study clearly suggests that genistein and daidzin enhance AChE activityin PC12 cells by binding to the ER; however, the actual mechanism of enhancement is not known.