Characterization of the {alpha}-helix region in domain 3 of the haemolytic lectin CEL-III: implications for self-oligomerization and haemolytic processes

CEL-III is a haemolytic lectin, which has two beta-trefoil domains (domains 1 and 2) and a beta-sheet-rich domain (domain 3). In domain 3 (residues 284-432), there is a hydrophobic region containing two alpha-helices (H8 and H9, residues 317-357) and a loop between them, in which alternate hydrophobic residues, especially Val residues, are present. To elucidate the role of the alpha-helix region in the haemolytic process, peptides corresponding to different parts of this region were synthesized and characterized. The peptides containing the sequence that corresponded to the loop and second alpha-helix (H9) showed the strongest antibacterial activity for Staphylococcus aureus and Bacillus subtilis through a marked permeabilization of the bacterial cell membrane. The recombinant glutathione S-transferase (GST)-fusion proteins containing domain 3 or the alpha-helix region peptide formed self-oligomers, whereas mutations in the alternate Val residues in the alpha-helix region lead to decreased oligomerization ability of the fusion proteins. These results suggest that the alpha-helix region, particularly its alternate Val residues are important for oligomerization of CEL-III in target cell membranes, which is also required for a subsequent haemolytic action.     

Functionally undefined gene, yggE, alleviates oxidative stress generated by monoamine oxidase in recombinant Escherichia coli

Real-time PCR analysis showed that yggE gene was about two and three times up-regulated in Escherichia coli cells exposed to UVA irradiation and thermal elevation, respectively, suggesting that this gene is responsive to physiological stress. The yggE gene was introduced into E. coli BL21 cells, together with a monoamine oxidase (MAO) gene as a model source for oxidative stress generation. The distribution of independently isolated transformants (two dozen isolates) was examined in terms of MAO activity and cell vitality. In the case of control strain expressing MAO alone, the largest number of transformants existed in the low range of MAO activity less than 2 units mg⁻¹ and the number significantly decreased at increased MAO activity. On the other hand, the distribution of MAO/YggE-coexpressing transformants shifted to higher MAO activity with frequent appearance in the activity range of 4–8 units mg⁻¹. The yggE gene product therefore has a possible function for alleviating the stress generated in the cells.     

Identification of novel ribonucleo-protein complexes from the brain-specific snoRNA MBII-52

Small nucleolar RNAs (snoRNAs) guide nucleotide modifications within ribosomal RNAs or spliceosomal RNAs by base-pairing to complementary regions within their RNA targets. The brain-specific snoRNA MBII-52 lacks such a complementarity to rRNAs or snRNAs, but instead has been reported to target the serotonin receptor 2C pre-mRNA, thereby regulating pre-mRNA editing and/or alternative splicing. To understand how the MBII-52 snoRNA might be involved in these regulatory processes, we isolated the MBII-52 snoRNP from total mouse brain by an antisense RNA affinity purification approach. Surprisingly, by mass spectrometry we identified 17 novel candidates for MBII-52 snoRNA binding proteins, which previously had not been reported to be associated with canonical snoRNAs. Among these, Nucleolin and ELAVL1 proteins were confirmed to independently and directly interact with the MBII-52 snoRNA by coimmunoprecipitation. Our findings suggest that the MBII-52 snoRNA assembles into novel RNA-protein complexes, distinct from canonical snoRNPs.     

Exclusive Male Egg Care and Determinants of Brooding Success in a Millipede

The evolution of exclusive male parental care is a major and controversial issue in behavioural ecology. Although arthropods practicing paternal care are thought to be key taxa for investigating this issue, few studies have attempted to clarify the selection factors associated with male behaviour and fitness consequences in arthropods. In the millipede Brachycybe nodulosa, males curl their bodies around egg masses on the undersides of decaying logs. Male‐removal experiments in the laboratory strongly suggest that males defend the eggs against fungal infection. Orphaned eggs were soon covered by hyphae and no eggs hatched, whereas almost all eggs brooded by males successfully hatched. The egg‐brooding males showed no aggressive responses when disturbed. Only some mature males bred in the field. Furthermore, the number of eggs brooded varied greatly among the males. Selected generalized linear models revealed that males with a wide seventh body segment, which possesses gonopods (genital legs), tended to succeed in brooding; and males with a wider body also obtained more eggs. Colony attributes had no significant effects on male brooding. We discuss the possible sexual selection mechanisms that could accomplish this pattern of brooding success among male B. nodulosa.     

Design of a New Energy‐Harvesting Electrochromic Window Based on an Organic Polymeric Dye,a Cobalt Couple,and PProDOT‐Me2

A new design for an energy‐harvesting electrochromic window (EH‐ECW) based on the fusion of two technologies, organic electrochromic windows and dye‐sensitized solar cells (DSSCs), is presented. Unlike other power‐generating smart windows, such as photoelectrochromic devices that are passive and only contain two states (i.e., a closed‐circuit colored state and an open‐circuit bleaching state), EH‐ECW allows active tuning of the transmittance by varying the applied potential and it functions as a photovoltaic cell based on a DSSC. The resulting device demonstrates a fast switching rate of 1 s in both the bleaching and coloring processes through the use of an electrochromic polymer as a counter electrode layer. To increase the transmittance of the device, a cobalt redox couple and a light‐colored, yet efficient, organic dye are used. The organic dye contains a polymeric structure that contributes to the high cyclic stability. The device exhibits a power conversion efficiency (PCE) of 4.5% (100 mW cm^‐2) under AM 1.5 irradiation, a change in transmittance of 34% upon applied potential, and shows only 3% degradation in the PCE after 5000 cycles.     

Altered redox status in Escherichia coli cells enhances pyruvate production in pH-adjusting culture with a fermenter

Improvements in pyruvate production process were examined using Escherichia coli BW25113?pta/pHfdh strain carrying the formate dehydrogenase gene of Mycobacterium vaccae to change the redox status of the cells. Glucose and formate concentrations, and oxygenation levels determined previously in a shake-flask culture were applied for pyruvate production in a 1 l fermenter. However, pyruvate was not produced under the examined conditions. Detailed pH measurements during the fermenter culture using CaCO₃ revealed that maintaining the pH value around 6.0 plays an important role in stabilizing the pyruvate accumulation. In the pH-adjusting culture around 6.0 with NaOH solution, the concentration and yield of pyruvate were 8.96 g l^?1 and 0.48 g pyruvate g glucose^?1, respectively, which were significantly higher than the values reported in the shake-flask culture (6.79 g l^?1 and 0.32 g pyruvate g glucose^?1).     

Overtime Work and Prevalence of Diabetes in Japanese Employees: Japan Epidemiology Collaboration on Occupational Health Study

Objective

Epidemiologic evidence on long working hour and diabetes has been conflicting. We examined the association between overtime work and prevalence of diabetes among Japanese workers.

Methods

The subjects were 40,861 employees (35,170 men and 5,691 women), aged 16 to 83 years, of 4 companies in Japan. Hours of overtime were assessed using self-reported questionnaires. Diabetes was defined as a fasting plasma glucose ≥126 mg/dl (7.0 mmol/l), hemoglobin A1c ≥6.5% (48 mmol/mol), or current use of anti-diabetic drug. Multiple logistic regression analysis was used to calculate odds ratio of diabetes for each category of overtime.

Results

After adjustment for age, sex, company, smoking, and BMI, there was a suggestion of U-shaped relationship between overtime work and prevalence of diabetes (P for quadratic trend = 0.07). Compared with those who worked <45 hours of overtime per month, the adjusted odds ratios (95% confidence interval) of diabetes were 0.86 (0.77–0.94), 0.69 (0.53–0.89), and 1.03 (0.72–1.46) for those who worked 45–79, 80–99, and ≥100 hours of overtime per month, respectively. In one company (n = 33,807), where other potential confounders including shift work, job position, type of department, alcohol consumption, sleep duration, leisure time physical activity, and family history of diabetes was additionally adjusted for, similar result was obtained (P for quadratic trend = 0.05).

Conclusions

Long hours of overtime work may not be associated with increased prevalence of diabetes among Japanese workers.     

Frequency of and Predictive Factors for Vascular Invasion after Radiofrequency Ablation for Hepatocellular Carcinoma

Background

Vascular invasion in patients with hepatocellular carcinoma (HCC) is representative of advanced disease with an extremely poor prognosis. The detailed course of its development has not been fully elucidated.

Methods

We enrolled 1057 consecutive patients with HCC who had been treated with curative intent by radiofrequency ablation (RFA) as an initial therapy from 1999 to 2008 at our department. We analyzed the incidence rate of and predictive factors for vascular invasion. The survival rate after detection of vascular invasion was also analyzed.

Results

During a mean follow-up period of 4.5 years, 6075 nodules including primary and recurrent lesions were treated by RFA. Vascular invasion was observed in 97 patients. The rate of vascular invasion associated with site of original RFA procedure was 0.66% on a nodule basis. The incidence rates of vascular invasion on a patient basis at 1, 3, and 5 years were 1.1%, 5.9%, and 10.4%, respectively. Univariate analysis revealed that tumor size, tumor number, alpha-fetoprotein (AFP), des-gamma-carboxy prothrombin (DCP), and Lens culinaris agglutinin-reactive fraction of alpha-fetoprotein were significant risk predictors of vascular invasion. In multivariate analysis, DCP was the most significant predictor for vascular invasion (compared with a DCP of ≤100 mAu/mL, the hazard ratio was 1.95 when DCP was 101–200 mAu/mL and 3.22 when DCP was >200 mAu/mL). The median survival time after development of vascular invasion was only 6 months.

Conclusion

Vascular invasion occurs during the clinical course of patients initially treated with curative intent. High-risk patients may be identified using tumor markers.     

Hemolytic Lectin CEL-III Heptamerizes via a Large Structural Transition from α-Helices to a β-Barrel during the Transmembrane Pore Formation Process

CEL-III is a hemolytic lectin isolated from the sea cucumber Cucumaria echinata. This lectin is composed of two carbohydrate-binding domains (domains 1 and 2) and one oligomerization domain (domain 3). After binding to the cell surface carbohydrate chains through domains 1 and 2, domain 3 self-associates to form transmembrane pores, leading to cell lysis or death, which resembles other pore-forming toxins of diverse organisms. To elucidate the pore formation mechanism of CEL-III, the crystal structure of the CEL-III oligomer was determined. The CEL-III oligomer has a heptameric structure with a long β-barrel as a transmembrane pore. This β-barrel is composed of 14 β-strands resulting from a large structural transition of α-helices accommodated in the interface between domains 1 and 2 and domain 3 in the monomeric structure, suggesting that the dissociation of these α-helices triggered their structural transition into a β-barrel. After heptamerization, domains 1 and 2 form a flat ring, in which all carbohydrate-binding sites remain bound to cell surface carbohydrate chains, stabilizing the transmembrane β-barrel in a position perpendicular to the plane of the lipid bilayer.