Thermodynamics of ribonuclease T1 denaturation.

Differential scanning calorimetry has been used to investigate the thermodynamics of denaturation of ribonuclease T1 as a function of pH over the pH range 2-10, and as a function of NaCl and MgCl2 concentration. At pH 7 in 30 mM PIPES buffer, the thermodynamic parameters are as follows: melting temperature, T1/2 = 48.9 +/- 0.1 degrees C; enthalpy change, delta H = 95.5 +/- 0.9 kcal mol-1; heat capacity change, delta Cp = 1.59 kcal mol-1 K-1; free energy change at 25 degrees C, delta G degrees (25 degrees C) = 5.6 kcal mol-1. Both T1/2 = 56.5 degrees C and delta H = 106.1 kcal mol-1 are maximal near pH 5. The conformational stability of ribonuclease T1 is increased by 3.0 kcal/mol in the presence of 0.6 M NaCl or 0.3 M MgCl2. This stabilization results mainly from the preferential binding of cations to the folded conformation of the protein. The estimates of the conformational stability of ribonuclease T1 from differential scanning calorimetry are shown to be in remarkably good agreement with estimates derived from an analysis of urea denaturation curves.     

Synthesis, chiral HPLC resolution and configuration assignment of 1-phenylglyceryl trinitrate stereomers

As an introductory study of in vitro vasodilating activity, the access to the four stereomers of 1-phenylglyceryl trinitrate is described using achiral and chiral chromatography. For semi-preparative separation of the enantiomers, a Chiralcel OD (250 x 10 mm, 10 microm) was used. Catalytic reduction leading to the corresponding stereomers of 1-phenylglycerol allowed absolute configuration assignments. The same methods were used for the separation and configuration assignment of the enantiomers of 3-phenylpropane-1,2-diyl dinitrate.     

Response of phytoplankton and bacteria to nutrients and zooplankton: a mesocosm experiment

Although both nutrient inputs and zooplankton grazing are importantto phytoplankton and bacteria in lakes, controversy surroundsthe relative importance of grazing pressure for these two groupsof organisms. For phytoplankton, the controversy revolves aroundwhether zooplankton grazers, especially large cladocerans likeDaphnia, can effectively reduce phytoplankton populations regardlessof nutrient conditions. For bacteria, little is known aboutthe balance between possible direct and indirect effects ofboth nutrients and zooplankton grazing. However, there is evidencethat bacteria may affect phytoplankton responses to nutrientsor zooplankton grazing through direct or apparent competition.We performed a mesocosm experiment to evaluate the relativeimportance of the effects of nutrients and zooplankton grazingfor phytoplankton and bacteria, and to determine whether bacteriamediate phytoplankton responses to these factors. The factorialdesign crossed two zooplankton treatments (unsieved and sieved)with four nutrient treatments (0, 0.5, 1.0 and 2.0 µgphosphorus (P) l^–1 day^–1 together with nitrogen(N) at a N:P ratio of 20:1 by weight). Weekly sieving with 300µm mesh reduced the average size of crustacean zooplanktonin the mesocosms, decreased the numbers and biomass of Daphnia,and increased the biomass of adult copepods. Nutrient enrichmentcaused significant increases in phytoplankton chlorophyll a(4–5x), bacterial abundance and production (1.3x and 1.6x,respectively), Daphnia (3x) and total zooplankton biomass (2x).Although both total phytoplankton chlorophyll a and chlorophylla in the <35 µm size fraction were significantly lowerin unsieved mesocosms than in sieved mesocosms, sieving hadno significant effect on bacterial abundance or production.There was no statistical interaction between nutrient and zooplanktontreatments for total phytoplankton biomass or bacterial abundance,although there were marginally significant interactions forphytoplankton biomass <35 µm and bacterial production.Our results do not support the hypothesis that large cladoceransbecome less effective grazers with enrichment; rather, the differencebetween phytoplankton biomass in sieved versus unsieved zooplanktontreatments increased across the gradient of nutrient additions.Furthermore, there was no evidence that bacteria buffered phytoplanktonresponses to enrichment by either sequestering P or affectingthe growth of zooplankton.     

Do You Really Want to Know? Elementary Music Personnel and Potential in Utah

This is the second of two articles reporting the results of a study by the author regarding the status of elementary music education in the state of Utah. This article focuses on the qualifications of Utah's elementary music teachers (music certified, elementary classroom certified, artists-in-residence, volunteers, and paraprofessionals) and the conditions under which they teach. Interactions among teacher qualifications and teaching status are explored. Paraprofessionals play a significant role in Utah's elementary music programs. While over 90 percent of elementary schools in the United States provide regular music instruction taught by certified music specialists, less than 10 percent of Utah's elementary population receives such instruction. Nearly half of the elementary students in the state receive no designated music instruction beyond that provided by their regular elementary classroom teacher. The influence of school funding, No Child Left Behind and other accountability measures, high-stakes testing, urban/rural populations, and leadership are highlighted. Policy considerations are discussed.     

Cutting edge: IL-4-induced protection of CD4+CD25- Th cells from CD4+CD25+ regulatory T cell-mediated suppression

CD4(+)CD25(+) T regulatory (Treg) cells are a CD4(+) T cell subset involved in the control of the immune response. In vitro, murine CD4(+)CD25(+) Treg cells inhibit CD4(+)CD25(-) Th cell proliferation induced by anti-CD3 mAb in the presence of APCs. The addition of IL-4 to cocultured cells inhibits CD4(+)CD25(+) Treg cell-mediated suppression. Since all cell types used in the coculture express the IL-4Ralpha chain, we used different combinations of CD4(+)CD25(-) Th cells, CD4(+)CD25(+) Treg cells, and APCs from wild-type IL-4Ralpha(+/+) or knockout IL-4Ralpha(-/-) mice. Results show that the engagement of the IL-4Ralpha chain on CD4(+)CD25(-) Th cells renders these cells resistant to suppression. Moreover, the addition of IL-4 promotes proliferation of IL-4Ralpha(+/+)CD4(+)CD25(+) Treg cells, which preserve full suppressive competence. These findings support an essential role of IL-4 signaling for CD4(+)CD25(-) Th cell activation and indicate that IL-4-induced proliferation of CD4(+)CD25(+) Treg cells is compatible with their suppressive activity.     

Free fatty acid-induced hepatic insulin resistance: a potential role for protein kinase C-delta

The mechanisms of the impairment in hepatic glucose metabolism induced by free fatty acids (FFAs) and the importance of FFA oxidation in these mechanisms remain unclear. FFA-induced peripheral insulin resistance has been linked to membrane translocation of novel protein kinase C (PKC) isoforms, but the role of PKC in hepatic insulin resistance has not been assessed. To investigate the biochemical pathways that are induced by FFA in the liver and their relation to glucose metabolism in vivo, we determined endogenous glucose production (EGP), the hepatic content of citrate (product of acetyl-CoA derived from FFA oxidation and oxaloacetate), and hepatic PKC isoform translocation after 2 and 7 h Intralipid + heparin (IH) or SAL in rats. Experiments were performed in the basal state and during hyperinsulinemic clamps (insulin infusion rate, 5 mU. kg(-1). min(-1)). IH increased EGP in the basal state (P < 0.001) and during hyperinsulinemia (P < 0.001) at 2 and 7 h. Also, 7-h infusion of IH induced resistance to the suppressive effect of insulin on EGP (P < 0.05). Glycerol infusion (resulting in plasma glycerol levels similar to IH infusion) did not have any effect on EGP. IH increased hepatic citrate content by twofold, independent of the insulin levels and the duration of IH infusion. IH induced hepatic PKC-delta translocation from the cytosolic to membrane fraction in all groups. PKC-delta translocation was greater at 7 compared with 2 h (P < 0.05). In conclusion, 1) increased FFA oxidation may contribute to the FFA-induced increase in EGP in the basal state and during hyperinsulinemia but is not associated with FFA-induced hepatic insulin resistance, and 2) the progressive insulin resistance induced by FFA in the liver is associated with a progressive increase in hepatic PKC-delta translocation.     

An unusual 5S rRNA, from Sulfolobus acidocaldarius, and its implications for a general 5S rRNA structure.

The nucleotide sequence of the 5S ribosomal RNA of the thermoacidophilic archaebacterium Sulfolobus acidocaldarius was determined. The high degree of evident secondary structure in the molecule has implications for the common higher order structure of other 5S rRNAs, both bacterial and eukaryotic.     

Assessment of spermatozoal function using dual fluorescent staining and flow cytometric analyses

Spermatozoa from bulls, boars, dogs, horses, mice, and men were examined using a fluorogenic stain consisting of the membrane-permeant substrate carboxyfluorescin diacetate (CFDA) and the relatively membrane-impermeant nuclear stain propidium iodide (PI). Three distinct populations of spermatozoa were discernible in samples from each species upon microscopic examination. Individual spermatozoa, presumed to be viable because of their motility, retained products of the fluorescein chromophore throughout the cell. A second population of spermatozoa in which the nuclei stained red with PI retained the green fluorescein fluorophore mainly in the acrosome. A third population, presumed to be degenerate spermatozoa, possessed only red fluorescent nuclei. These populations were quantified using dual parameter flow cytometry in 14 samples of cryopreserved bovine spermatozoa for which fertility and seminal quality data were available. Flow cytometric analyses were highly correlated with other seminal quality measurements. Sequential flow cytometric analyses provided the ability to rapidly quantitate changes in specific fluorescently stained populations. The ability to make rapid quantitative measurements should allow development of new and presumably more reliable information on the functional aspects of spermatozoa.     

Non‐additive stabilization by halogenated amino acids reveals protein plasticity on a sub‐angstrom scale

It has been a long‐standing goal to understand the structure‐stability relationship of proteins, as optimal stability is essential for protein function and highly desirable for protein therapeutics. Halogenation has emerged as a minimally invasive strategy to probe the physical characteristics of proteins in solution, as well as enhance the structural stabilities of proteins for therapeutic applications. Although advances in synthetic chemistry and genetic code expansion have allowed for the rapid synthesis of proteins with diverse chemical sequences, much remains to be learned regarding the impact of these mutations on their structural integrity. In this contribution, we present a systematic study of three well‐folded model protein systems, in which their structural stabilities are assessed in response to various hydrogen‐to‐halogen atom mutations. Halogenation allows for the perturbation of proteins on a sub‐angstrom scale, offering unprecedented precision of protein engineering. The thermodynamic results from these model systems reveal that in certain cases, proteins can display modest steric tolerance to halogenation, yielding non‐additive consequences to protein stability. The observed sub‐angstrom sensitivity of protein stability highlights the delicate arrangement of a folded protein core structure. The stability data of various halogenated proteins presented herein should also provide guidelines for using halogenation as a strategy to improve the stability of protein therapeutics.     

Effects of climate change on the delivery of soil‐mediated ecosystem services within the primary sector in temperate ecosystems: a review and New Zealand case study

Future human well‐being under climate change depends on the ongoing delivery of food, fibre and wood from the land‐based primary sector. The ability to deliver these provisioning services depends on soil‐based ecosystem services (e.g. carbon, nutrient and water cycling and storage), yet we lack an in‐depth understanding of the likely response of soil‐based ecosystem services to climate change. We review the current knowledge on this topic for temperate ecosystems, focusing on mechanisms that are likely to underpin differences in climate change responses between four primary sector systems: cropping, intensive grazing, extensive grazing and plantation forestry. We then illustrate how our findings can be applied to assess service delivery under climate change in a specific region, using New Zealand as an example system. Differences in the climate change responses of carbon and nutrient‐related services between systems will largely be driven by whether they are reliant on externally added or internally cycled nutrients, the extent to which plant communities could influence responses, and variation in vulnerability to erosion. The ability of soils to regulate water under climate change will mostly be driven by changes in rainfall, but can be influenced by different primary sector systems' vulnerability to soil water repellency and differences in evapotranspiration rates. These changes in regulating services resulted in different potentials for increased biomass production across systems, with intensively managed systems being the most likely to benefit from climate change. Quantitative prediction of net effects of climate change on soil ecosystem services remains a challenge, in part due to knowledge gaps, but also due to the complex interactions between different aspects of climate change. Despite this challenge, it is critical to gain the information required to make such predictions as robust as possible given the fundamental role of soils in supporting human well‐being.