Crystallization of Adenylylsulfate Reductase from Desulfovibrio gigas: A Strategy Based on Controlled Protein Oligomerization

Adenylylsulfate reductase (adenosine 5′-phosphosulfate reductase, APS reductase or APSR, E.C.1.8.99.2) catalyzes the conversion of APS to sulfite in dissimilatory sulfate reduction. APSR was isolated and purified directly from massive anaerobically grown Desulfovibrio gigas, a strict anaerobe, for structure and function investigation. Oligomerization of APSR to form dimers–α_2β_2, tetramers–α_4β_4, hexamers–α_6β_6, and larger oligomers was observed during purification of the protein. Dynamic light scattering and ultracentrifugation revealed that the addition of adenosine monophosphate (AMP) or adenosine 5′-phosphosulfate (APS) disrupts the oligomerization, indicating that AMP or APS binding to the APSR dissociates the inactive hexamers into functional dimers. Treatment of APSR with β-mercaptoethanol decreased the enzyme size from a hexamer to a dimer, probably by disrupting the disulfide Cys156—Cys162 toward the C-terminus of the β-subunit. Alignment of the APSR sequences from D. gigas and A. fulgidus revealed the largest differences in this region of the β-subunit, with the D. gigas APSR containing 16 additional amino acids with the Cys156—Cys162 disulfide. Studies in a pH gradient showed that the diameter of the APSR decreased progressively with acidic pH. To crystallize the APSR for structure determination, we optimized conditions to generate a homogeneous and stable form of APSR by combining dynamic light scattering, ultracentrifugation, and electron paramagnetic resonance methods to analyze the various oligomeric states of the enzyme in varied environments

Earthquake Doublet Sequences: Evidence of Static Triggering in the Strong Convergent Zones of Taiwan

Three earthquake sequences, each with two main earthquakes of almost the same magnitudes (ML from 5.9 to 7.0 with differences less than 0.1), have recently been observed in Taiwan. The two largest earthquakes in each sequence occurred with a short delay time between them, were strikingly similar in terms of magnitude, location and/or focal mechanism and are referred to as doublets. They were markedly distinct from typical single mainshock sequences. Our estimated static stress field generated by the first shock in the doublet shows that the second shock and most of their aftershocks were located within a region where static stress increased substantially. Thus, a possible explanation for earthquake doublet is that seismic energy for each shock had accumulated independently within adjacent crustal volumes, separated by an asperity, and that the second shock is triggered by the increased static stress after the first one. An important implication of doublet sequence is that works by emergency response teams after the first earthquake could be made more hazardous by the second

Ray Tracing Simulation in Nonspherically Symmetric Atmosphere for GPS Radio Occultation

A three-dimensional ray tracing model with aiming algorithms for global positioning system (GPS) signal is proposed to make simulations conform to the realistic radio occultation (RO) signal propagation. The two aiming algorithms used in this study ensure the initial and end point ray trajectories are located in the prescribed region. In past studies, the ray tracing techniques applied to the RO signal simulation usually assumed a spherically symmetrical atmosphere for simplicity. The exact GPS and low earth orbit (LEO) satellite locations are not considered in the simulation. These two assumptions make the simulation unrealistic for GPS signal propagation in the RO technique. In the proposed model, the shape of the earth is assumed as an ellipse. The information from European Centre for Medium-Range Weather Forecasts (ECMWF) analysis is used to setup the atmosphere in the simulation. Two aiming algorithms are developed to determine the initial signal propagating direction to make the simulated signal start from the prescribed GPS satellite position and end in the close vicinity of the LEO satellite position. An ideal spherical symmetric atmospheric structure is used to verify the ray tracing model. The fractional difference between real and simulated refractivity results is less than 0.1%. Otherwise, the GPS and LEO satellite position in the Formosat-3/COSMIC observation and the ECMWF analysis, considering the earth¡¦s flattening, is also used to verify the aiming algorithms. All of the simulated signals end in close vicinity to the LEO satellite position in the simulation results

A Comparison of Food-grade Folium mori Extract and 1-Deoxynojirimycin for Glycemic Control and Renal Function in Streptozotocin-induced Diabetic Rats

ABSTRACTFolium mori (桑葉 Sāng Yè, leaf of Morus alba L.; FM) is known to possess hypoglycemic effects, and 1-deoxynojirimycin (1-DNJ) has been proposed as an important functional compound in FM. However, the hypoglycemic activity of purified 1-DNJ has been rarely studied. It is also not known how FM and 1-DNJ affect the development of DM nephropathy. This study compared the antidiabetic effect of a commercial FM product with that of purified 1-DNJ in streptozotocin-induced diabetic rats. Seven days after induction, the diabetic rats were gavaged with FM (1, 3, 10, and 30mg/kg/day), 1-DNJ (30mg/kg/day), or vehicle (distilled deionized water; 2ml/kg/day) for 7days. All doses of FM ameliorated fasting and post-prandial blood glucose concomitantly with an increase in peripheral and pancreatic levels of insulin and improved homeostasis model assessment (HOMA-IR) in diabetic rats in a dose-dependent manner. Increased thiobarbituric acid reactive substances (TBARS) and nitrate/nitrite levels in the kidney, liver, and muscle of diabetic rats were reversed by all doses of FM. The renal function of the diabetic rats was normalized by all doses of FM, while blood pressure changes were reversed by FM at doses of 3mg/kg and above. Moreover, most of the above-mentioned parameters were improved by FM at doses of 3mg/kg and above to a similar extent as that of 1-DNJ. The results showed superior antidiabetic potential of the commercial FM product for glycemic control and protection against the development of diabetic nephropathy

Gender Difference in the Relationship of Albuminuria and Arterial Stiffness in Chinese Adults - a 6.6-Year Follow-Up Longitudinal Study

Background/Aims: Brachial–ankle pulse wave velocity (baPWV) reflects the stiffness of muscular arteries. Albuminuria is recognized as a marker of vascular dysfunction. We assessed the association between arterial stiffness and albuminuria in a population-based longitudinal study. Methods: 1116 adults aged ≥ 40 years in the Taichung Community Health Study (TCHS) in 2004 attended a follow-up visit in 2011. Albuminuria was defined as an urinary albumin-to-creatinine ratio (UACR) ≥ 30 mg/g. Arterial stiffness was defined as BaPWV ≥ 1540 cm/sec in males and BaPWV ≥ 1480 cm/sec in females, respectively. ∆baPWV was calculated as baPWV at follow-up minus baPWV at baseline, while ∆UACR was calculated as UACR at follow-up minus UACR at baseline. Multiple linear and logistic regression analyses were used to explore the relationship between albuminuria and arterial stiffness. Results: Among 652 subjects without arterial stiffness at baseline, 209 (32%) subjects developed incident arterial stiffness after an average of 6.6 years. In male subjects, baseline albuminuria was associated with development of arterial stiffness (odds ratio: 4.47, 95% confidence interval [CI]: 1.04–19.31) and ∆baPWV was modestly positively associated with ∆UACR. Conclusion: Our results indicated that male adults with albuminuria had an increased risk for developing arterial stiffness

Areca Users in Combination with Tobacco and Alcohol Use Are Associated with Younger Age of Diagnosed Esophageal Cancer in Taiwanese Men

BACKGROUND: Whether the habitual use of substances (tobacco, alcohol, or areca nut (seed of the Areca palm)) can affect the age of esophageal squamous cell carcinoma (ESCC) presentation has rarely been examined. METHODS: The study subjects were those who were males and the first time to be diagnosed as ESCC (ICD-9 150) and who visited any of three medical centers in Taiwan between 2000 and 2009. A standardized questionnaire was used to collect substance uses and other variables. RESULTS: Mean age (±SD) at presentation of ESCC was 59.2 (±11.3) years in a total of 668 cases. After adjusting for other covariates, alcohol drinkers were 3.58 years younger to have ESCC than non-drinkers (p = 0.002). A similar result was found among areca chewers, who were 6.34 years younger to have ESCC than non-chewers (p<0.0001), but not among cigarette smokers (p = 0.10). When compared to the group using 0-1 substances, subjects using both cigarettes and alcohol were nearly 3 years younger to contract ESCC. Furthermore, those who use areca plus another substance were 7-8 years younger. Subjects using all three substances had the greatest age difference, 9.20 years younger (p<0.0001), compared to the comparison group. CONCLUSION: Our findings suggest that habitually consuming tobacco, alcohol, and areca nut can influence the age-onset of ESCC. Since the development of ESCC is insidious and life-threatening, our observation is worthy to be reconfirmed in the large-scale and long-term follow-up prospective cohort studies to recommend the screening strategy of this disease

Assembling a cellulase cocktail and a cellodextrin transporter into a yeast host for CBP ethanol production

Background: Many microorganisms possess enzymes that can efficiently degrade lignocellulosic materials, but donot have the capability to produce a large amount of ethanol. Thus, attempts have been made to transform suchenzymes into fermentative microbes to serve as hosts for ethanol production. However, an efficient host for aconsolidated bioprocess (CBP) remains to be found. For this purpose, a synthetic biology technique that cantransform multiple genes into a genome is instrumental. Moreover, a strategy to select cellulases that interactsynergistically is needed.Results: To engineer a yeast for CBP bio-ethanol production, a synthetic biology technique, called “promoter-basedgene assembly and simultaneous overexpression” (PGASO), that can simultaneously transform and express multiplegenes in a kefir yeast, Kluyveromyces marxianus KY3, was recently developed. To formulate an efficient cellulasecocktail, a filter-paper-activity assay for selecting heterologous cellulolytic enzymes was established in this study andused to select five cellulase genes, including two cellobiohydrolases, two endo-β-1,4-glucanases and onebeta-glucosidase genes from different fungi. In addition, a fungal cellodextrin transporter gene was chosen totransport cellodextrin into the cytoplasm. These six genes plus a selection marker gene were one-step assembledinto the KY3 genome using PGASO. Our experimental data showed that the recombinant strain KR7 could expressthe five heterologous cellulase genes and that KR7 could convert crystalline cellulose into ethanol.Conclusion: Seven heterologous genes, including five cellulases, a cellodextrin transporter and a selection marker,were simultaneously transformed into the KY3 genome to derive a new strain, KR7, which could directly convertcellulose to ethanol. The present study demonstrates the potential of our strategy of combining a cocktailformulation protocol and a synthetic biology technique to develop a designer yeast host