Changes in the origin and quality of soil organic matter after pasture introduction in Rondônia (Brazil)

We examined the effects of the conversion of tropical forest to pasture on soil organic matter (SOM) origin and quality along a chronosequence of sites, including a primary forest and six pastures. Bulk soil samples received a physical size-fractionation treatment to assess the contribution of each compartment to total SOM pool. Besides a general increase in total C and N stocks along the chronosequence, we observed a reduction of the relative contribution of the coarser fractions to total soil C content, and an increased concentration in the finer fractions. The origin of the C in each size fraction was established from measurements of¹³C abundance. After 80 years about 93% of the C in the least humified fraction of the top 10 cm of soil was of pasture origin, while in the most humified it was 82%. Chemical analyses indicated that the fine silt and coarse clay fractions contained the most refractory carbon.     

A proteomics approach to cloning fenestrin from the nuclear exchange junction of tetrahymena

ABSTRACT. We set out to find the " fenestrin " gene, a gene whose protein is associated with numerous cellular apertures, including the nuclear exchange junction in mating Tetrahymena thermophila . First we developed protocols for imaging and isolating intact nuclear exchange junctions from conjugating cells. Proteins from these junctions were purified using SDS-PAGE, subjected to limited proteolysis, and precise molecular weights were determined by mass spectrometry. Using Protein Prospector^® software and the published Tetrahymena Genome Database, genes for 15 of the most abundant proteins found in our extracts were identified. The most promising candidate was cloned by PCR, fused to yellow fluorescent protein (YFP), and placed under the control of an inducible metallothionein promoter. YFP-localization within live Tetrahymena transformants strongly suggested that one of these genes encoded the fenestrin protein, a result that was subsequently confirmed by Western blotting.     

Biotechnological approach to the synthesis of 9alpha-hydroxylated steroids

The steroid 9alpha-hydroxylase gene has been cloned from Mycobacterium smegmatis into Escherichia coli BL21. Progesterone added to bioreactors was subjected to in vivo transformation into 9alpha-hydroxyprogesterone. In 7 days, 43.6 mg 9alpha-hydroxyprogesterone was formed from 53.8 mg/L progesterone. The enzyme also has shown evidence of processing 4-androstene-3,17-dione in vivo. An extensive analytical method development, including LLE, HPLC-DAD, MS, and NMR was performed to verify the product and to enable a quantitative analysis. Protocols for analytical and preparative separation have been developed, using binaphtol as internal standard. Both the growth pattern and the bioconversion rate were unaffected by the presence of binaphtol in the bioreactor. The enzyme was purified by immobilised metal affinity and ion exchange chromatography, resulting in low in vitro activity.     

Detection and analysis of membrane interactions by a biomimetic colorimetric lipid/polydiacetylene assay

We describe applications of a colorimetric assay based on supramolecular assemblies of lipid-polydiacetylene vesicles for analysis and screening of membrane interactions of lipophilic enzymes, peptides, and ions and for study of the effects of lipid composition upon membrane properties. The lipid-polymer aggregates undergo visible and quantifiable blue-to-red transitions following interfacial interactions and perturbation by varied biochemical processes. Specifically, we show that the colorimetric assay can be tuned for selective detection of enzymes reacting with different lipid species. The experiments also demonstrate that the lipid/polymer platform facilitates screening of peptide-membrane interactions in multicomponent mixtures. The colorimetric vesicles can incorporate lipid species from different cellular sources facilitating analysis of the contribution of molecular components to membrane properties and lipid interactions.     

Continuous measurement of changes in intracellular calcium concentration in mouse splenic T cells attached to a glass substrate

Mitogen- and isoproterenol-induced changes of [Ca²⁺]_i in T cells attached to a glass substrate were examined. Murine (C57BL/6) splenic T cells were attached to coverslips or 35-mm dishes (MatTek) precoated with Cell Tak® (3.5 µg/cm²). The cells were then loaded with fluorescent dye (2 µg/ml of fura2-AM or fluo3-AM) and changes in [Ca²⁺]_i in a population of cells (using a spectrofluorometer) or in single cells (using a confocal microscope) were measured during continuous superfusion. Population measurements of [Ca²⁺]_i demonstrated that concanavalin A (Con A, 2 or 5 µg/ml) caused an increase in [Ca²⁺]_i that rose to a peak and then declined to a steady state. The concentration-response relationship (0.05–5 µg/ml) had an EC₅₀ of 0.3 µg/ml. Isoproterenol decreased the Con A-induced elevation of steady state [Ca²⁺]_i. In single cell studies, the increase in [Ca²⁺]_i in response to Con A typically occurred in about 50% of the cells in a microscope field, and the delay before activation varied among cells. Taken together, these data demonstrate that Cell Tak® can be used to attach T cells to glass coverslips and will be useful for the study of signaling mechanisms in T cells.     

“Entombed Pollen”: A new condition in honey bee colonies associated with increased risk of colony mortality

Here we describe a new phenomenon, entombed pollen, which is highly associated with increased colony mortality. Entombed pollen is sunken, capped cells amidst “normal”, uncapped cells of stored pollen, and some of the pollen contained within these cells is brick red in color. There appears to be a lack of microbial agents in the pollen, and larvae and adult bees do not have an increased rate of mortality when they are fed diets supplemented with entombed pollen in vitro, suggesting that the pollen itself is not directly responsible for increased colony mortality. However, the increased incidence of entombed pollen in reused wax comb suggests that there is a transmittable factor common to the phenomenon and colony mortality. In addition, there were elevated pesticide levels, notably of the fungicide chlorothalonil, in entombed pollen. Additional studies are needed to determine if there is a causal relationship between entombed pollen, chemical residues, and colony mortality.