Ordered and dynamic assembly of single spliceosomes

The spliceosome is the complex macromolecular machine responsible for removing introns from precursors to messenger RNAs (pre-mRNAs). We combined yeast genetic engineering, chemical biology, and multiwavelength fluorescence microscopy to follow assembly of single spliceosomes in real time in whole-cell extracts. We find that individual spliceosomal subcomplexes associate with pre-mRNA sequentially via an ordered pathway to yield functional spliceosomes and that association of every subcomplex is reversible. Further, early subcomplex binding events do not fully commit a pre-mRNA to splicing; rather, commitment increases as assembly proceeds. These findings have important implications for the regulation of alternative splicing. This experimental strategy should prove widely useful for mechanistic analysis of other macromolecular machines in environments approaching the complexity of living cells.     

Evaluation of spectrophotometric and HPLC methods for shikimic acid determination in plants: models in glyphosate-resistant and -susceptible crops

Endogenous shikimic acid determinations are routinely used to assess the efficacy of glyphosate in plants. Numerous analytical methods exist in the public domain for the detection of shikimic acid, yet the most commonly cited comprise spectrophotometric and high-pressure liquid chromatography (HPLC) methods. This paper compares an HPLC and two spectrophotometric methods (Spec 1 and Spec 2) and assesses the effectiveness in the detection of shikimic acid in the tissues of glyphosate-treated plants. Furthermore, the study evaluates the versatility of two acid-based shikimic acid extraction methods and assesses the longevity of plant extract samples under different storage conditions. Finally, Spec 1 and Spec 2 are further characterized with respect to (1) the capacity to discern between shikimic acid and chemically related alicyclic hydroxy acids, (2) the stability of the chromophore (t1/2), (3) the detection limits, and (4) the cost and simplicity of undertaking the analytical procedure. Overall, spectrophotometric methods were more cost-effective and simpler to execute yet provided a narrower detection limit compared to HPLC. All three methods were specific to shikimic acid and detected the compound in the tissues of glyphosate-susceptible crops, increasing exponentially in concentration within 24 h of glyphosate application and plateauing at approximately 72 h. Spec 1 estimated more shikimic acid in identical plant extract samples compared to Spec 2 and, likewise, HPLC detection was more effective than spectrophotometric determinations. Given the unprecedented global adoption of glyphosate-resistant crops and concomitant use of glyphosate, an effective and accurate assessment of glyphosate efficacy is important. Endogenous shikimic acid determinations are instrumental in corroborating the efficacy of glyphosate and therefore have numerous applications in herbicide research and related areas of science as well as resolving many commercial issues as a consequence of glyphosate utilization.     

Cinnamon polyphenol extract regulates tristetraprolin and related gene expression in mouse adipocytes

Cinnamon (Cinnamomum verum) has been widely used in spices, flavoring agents, and preservatives. Cinnamon polyphenol extract (CPE) may be important in the alleviation of chronic diseases, but the molecular evidence is not substantial. Tristetraprolin (TTP) family proteins have anti-inflammatory effects through the destabilization of pro-inflammatory mRNAs. TTP expression is reduced in fats of obese people with metabolic syndrome and brains of suicide victims. This study used quantitative real-time PCR to explore the effects of CPE on the regulation of TTP, VEGF, and related gene expression in mouse 3T3-L1 adipocytes. CPE (100 μg/mL) increased TTP mRNA levels by up to 10-fold, and this stimulation was sustained over 16 h. The levels of VEGF mRNA, a putative target of TTP, were decreased 40-50% by CPE. It also affected the expression of other genes coding for ZFP36L1 and ZFP36L3 (TTP homologues), GM-CSF, COX2, IL6, APP, G-CSF, and PAI1. This study demonstrated that CPE rapidly induces TTP mRNA and reduces VEGF mRNA and affects the expression of a number of other genes in the cultured adipocytes.     

Uterine adenocarcinoma with abdominal metastases in an ovariohysterectomised cat

SURGICAL FINDINGS: an adenocarcinoma of the uterine stump with abdominal metastases is described in a 12.5-year-old incompletely ovariohysterectomised domestic shorthair cat. At the time of presentation, the adenocarcinoma had metastasised to the right perirenal lymph node, the abdominal aorta and the right ureter, resulting in the formation of a large cystic structure. This had compressed and displaced surrounding structures, including the abdominal vena cava and the right kidney, and formed multiple adhesions to the body wall and adjacent abdominal structures. Metastatic extension to the aorta had resulted in its regression into a 2 mm diameter non-pulsatile vessel. PRACTICAL RELEVANCE: only one case of uterine adenocarcinoma has previously been reported in an ovariohysterectomised cat. As such, this represents a very unusual and severe complication following an incomplete ovariohysterectomy. Invasion of the tumour tissue into surrounding structures created further complications.     

Determination of angiotensin I-converting enzyme activity in equine blood: lack of agreement between methods of analysis

Angiotensin-I converting enzyme (ACE) is a key regulator of blood pressure, electrolytes and fluid homeostasis through conversion of angiotensin I into angiotensin II. Recently, a genetic polymorphism of the ACE gene, which accounts for 47% of the variation of ACE activity in blood, has been advocated as a biomarker of athletic aptitude. Different methods of analysis and determination of ACE activity in plasma have been used in human and equine research without a consensus of a "gold standard" method. Different methods have often been used interchangeably or cited as being comparable in the existing literature; however, the actual agreement between assays has not been investigated. Therefore, in this study, we evaluated the level of agreement between three different assays using equine plasma obtained from 29 horses. Two spectrophotometric assays using Furylacryloyl-phenylalanyl-glycyl-glycine as substrate and one fluorimetric assay utilizing o-aminobenzoic acid-FRK-(Dnp)P-OH were employed. The results revealed that the measurements from the different assays were not in agreement, indicating that the methods should not be used interchangeably for measurement of equine ACE activity. Rather, a single method of analysis should be adopted to achieve comparable results and critical appraisal of the literature is needed when attempting to compare results obtained from different assays.     

Evaluation of hematology and serum biochemistry of cold-stunned green sea turtles (Chelonia mydas) in North Carolina, U.S.A

Hypothermia or cold-stunning is a condition in which the body temperature of an animal decreases below normal physiologic range and which has been linked to severe morbidity in sea turtles. Reports have focused on the physiologic changes caused by cold-stunning in Kemp's Ridley sea turtles (Lepidochelys kempii) and loggerhead sea turtles (Caretta caretta), but few have evaluated the green sea turtle (Chelonia mydas). This study evaluated hematologic and serum biochemical profiles of cold-stunned green sea turtles in North Carolina, USA. When compared with healthy, free-ranging juvenile green turtles from the same region, cold-stunned turtles exhibited hypoglycemia, hypocalcemia (both total and ionized calcium), hyponatremia, hypokalemia, hypoproteinemia, hypoalbuminemia, hyperphosphatemia, and elevations in uric acid and blood urea nitrogen. These findings contrast with some previously reported changes in cold-stunned Kemp's Ridley and loggerhead sea turtles. These results emphasize the importance of basing therapeutic regimens on biochemical analyses in cold-stunned sea turtles.     

Alkalinity production in epilimnetic sediments: Acidic and non-acidic lakes

Interstitial water profiles in epilimnetic sediments of lakes with varying water column alkalinities were collected to assess the origin and importance of sedimentary alkalinity in freshwater lakes. Release of Ca²⁺ and NH₄ ⁺, and consumption of SO₄ ^? are the most important contributors to alkalinity production m sediments of non-acidic lakes. In acidic lakes, Fe²⁺ and Mn²⁺ replace Ca²⁺ as the dominant cation contributors to alkalinity production. The sedimentary alkalinity flux is an important component of the acid neutralizing capacity of freshwater lakes. However, the presence of large alkalinity gradients in sediment porewaters does not necessarily indicate a large source of alkalinity for the lake, as a significant portion of the alkalinity iu associated with the formation of Fe²⁺, Mn²⁺ and NH₄ ⁺ Oxidation of Fe²⁺ and Mn²⁺ at the anoxic-oxic interface and biological removal of NH₄ ⁺ in the overlying water column results in consumption of the co-diffusing alkalinity.     

Carbon assimilation and microbial activity in soil

In order to characterise the term microbial ?activity”? three different microbial populations belonging to a luvisol (I), a phaeozem (II) and a rendzina (III) were used for studying kinetic parameters such as substrate affinity, growth rate, yield and turnover time and the metabolic quotient of basal respiration. Glucose was used as a carbon source. Specific growth rate values (μ) varied between 0.0037 and 0.015 h^?1 depending on soil type and glucose concentration and were far below the potential μ_max. The calculated turnover time was 3–11 days, respectively. The yield coefficient was in the range between 0.37 and 0.53. The maximal uptake rate of glucose–C of soil population (II) was 0.041 g C g^?1 biomass-C h^?1. The determined affinity constant (K_m) was 57 μg C g^?1 soil. The affinity to glucose was higher for the glucose-mediated CO₂ evolution with K_m values of 15.2 and 17.5 than for the glucose uptake system itself. The observed qCO₂ values of the basal respiration at temperature increments from 0 to 45° C were almost identical for the soils (I) and (II). The calulated Q₁₀ lay in the range between 1.4 and 2.0.     

Methods to evaluate pesticide damage to the biomass of the soil microflora

Respiratory methods to estimate the amount of C in the soil microbial biomass and the relative contributions of procaryotes and eucaryotes to the biomass were used to evaluate the influence of pesticides on the soil microflora. Experiments were conducted with 5 and 50 μg·g^?1 of three fungicides, captan, thiram and verdasan. At 5 μg·g^?1 they caused significant decreases (40%) in the biomass; the organomercury fungicide verdasan also caused a shift from fungal to bacterial dominance. Within 8 days, biomass in captan- and thiram-amended soils had recovered to that of the controls. Although the fungal to bacterial balance was restored in verdasan-amended soils, biomass recovery was not complete. At 50 μg·g^?1 the fungicides caused long-term decreases in the biomass and altered the relative proportions of the bacterial and fungal populations. Verdasan had the greatest effect on soil microbial biomass and composition.