Shifts in allochthonous input and autochthonous production in streams along an agricultural land-use gradient

Relative contributions of allochthonous inputs and autochthonous production vary depending on terrestrial land use and biome. Terrestrially derived organic matter and in-stream primary production were measured in 12 headwater streams along an agricultural land-use gradient. Streams were examined to see how carbon (C) supply shifts from forested streams receiving primarily terrestrially derived C to agricultural streams, which may rely primarily on C derived from algal productivity. We measured allochthonous input, chlorophyll a concentration, and periphyton biomass in each stream, and whole-stream metabolism in six streams. Our results suggest a threshold between moderate- and heavy-agriculture land uses in which terrestrially derived C is replaced by in-stream algal productivity as the primary C source for aquatic consumers. A shift from allochthonous to autochthonous production was not evident in all heavy-agriculture streams, and only occurred in heavy-agriculture streams not impacted by livestock grazing. We then compared our findings to rates of allochthonous input and GPP in streams with minimal human influences in multiple biomes to assess how land-use practices influence C sources to stream ecosystems. The proportion of C derived from allochthonous versus autochthonous sources to heavy-agriculture streams was most similar to grassland and desert streams, while C sources to forested, light-, and moderate-agriculture streams were more similar to deciduous and montane coniferous forest streams. We show that C source to streams is dependent on land use, terrestrial biome, and degradation of in-stream conditions. Further, we suggest that within a biome there seems to be a compensation such that total C input is nearly equal whether it is from allochthonous or autochthonous sources.     

A Staining Combination for Phloem and Contiguous Tissues

A technic is described for producing critically stained preparations of phloem tissue. The preparations promise to be relatively stable. Sections of fixed unembedded or of embedded (paraffin or celloidin) phloem, cambium, and xylem are (1) stained in Foster's tannic acid-ferric chloride combination; (2) treated with 1% NaHCOg in 25% or 50% ethyl alcohol for 30 minutes; (3) stained in a saturated solution of lacmoid (made alkaline by adding a few ml. of 1% NaHCO₃ in 25% alcohol) for 12 to 18 hours; (4) dehydrated and cleared in a series composed of 1% solution of NaHCO_s in 50% ethyl alcohol, 80%, 95%, and absolute alcohol, equal proportions of absolute alcohol, clove oil, and xylene, and finally pure xylene; and (5) mounted in a neutral resin. Callose and lignified secondary walls are blue or blue-green in color, cellulose walls and stainable protoplasmic contents are generally light brown. The technic has been successful with sections from 5 to 40μ in thickness, and the staining has been satisfactory for both color and black and white photomicrography.     

Calculation of pK(a) in proteins with the microenvironment modulated-screened coulomb potential

The MM-SCP has been applied to predict pK(a) values of titratable residues in wild type and mutants of staphylococcal nuclease (SNase). The calculations were based on crystal structures made available by the Garcia-Moreno Laboratory. In the mutants, mostly deeply buried hydrophobic residues were replaced with ionizable residues, and thus their pK(a) values could be measured and calculated using various methods. The data set used here consisted of a set of WT SNase for which His pK(a) at several ionic strengths had been measured, a set of mutants for which measured pK(a) were available and a set of 11 mutants for which the measured pK(a) were not known at the time of calculation. For this latter set, blind predictions were submitted to the protein pK(a) cooperative, 2009 workshop at Telluride, where the results of the blind predictions were discussed (the RMSD of the submitted set was 1.10 pH units). The calculations on the structures with known pK(a) indicated that in addition to weaknesses of the method, structural issues were observed that led to larger errors (>1) in pK(a) predictions. For example, different crystallographic conditions or steric clashes can lead to differences in the local environment around the titratable residue, which can produce large differences in the calculated pK(a) . To gain further insight into the reliability of the MM-SCP, pK(a) of an extended set of 54 proteins belonging to several structural classes were carried out. Here some initial results from this study are reported to help place the SNase results in the appropriate context.     

Identification of a sporozoite-specific antigen from Toxoplasma gondii

Reduction of risk for human and food animal infection with Toxoplasma gondii is hampered by the lack of epidemiological data documenting the predominant routes of infection (oocyst vs. tissue cyst consumption) in horizontally transmitted toxoplasmosis. Existing serological assays can determine previous exposure to the parasite, but not the route of infection. We have used difference gel electrophoresis, in combination with tandem mass spectroscopy and Western blot, to identify a sporozoite-specific protein (T. gondii embryogenesis-related protein [TgERP]), which elicited antibody and differentiated oocyst- versus tissue cyst-induced infection in pigs and mice. The recombinant protein was selected from a cDNA library constructed from T. gondii sporozoites; this protein was used in Western blots and probed with sera from T. gondii -infected humans. Serum antibody to TgERP was detected in humans within 6-8 mo of initial oocyst-acquired infection. Of 163 individuals in the acute stage of infection (anti- T. gondii IgM detected in sera, or < 30 in the IgG avidity test), 103 (63.2%) had detectable antibodies that reacted with TgERP. Of 176 individuals with unknown infection route and in the chronic stage of infection (no anti- T. gondii IgM detected in sera, or > 30 in the IgG avidity test), antibody to TgERP was detected in 31 (17.6%). None of the 132 uninfected individuals tested had detectable antibody to TgERP. These data suggest that TgERP may be useful in detecting exposure to sporozoites in early T. gondii infection and implicates oocysts as the agent of infection.     

A structural approach reveals how neighbouring C2H2 zinc fingers influence DNA binding specificity

Development of an accurate protein–DNA recognition code that can predict DNA specificity from protein sequence is a central problem in biology. C₂H₂ zinc fingers constitute by far the largest family of DNA binding domains and their binding specificity has been studied intensively. However, despite decades of research, accurate prediction of DNA specificity remains elusive. A major obstacle is thought to be the inability of current methods to account for the influence of neighbouring domains. Here we show that this problem can be addressed using a structural approach: we build structural models for all C₂H₂-ZF–DNA complexes with known binding motifs and find six distinct binding modes. Each mode changes the orientation of specificity residues with respect to the DNA, thereby modulating base preference. Most importantly, the structural analysis shows that residues at the domain interface strongly and predictably influence the binding mode, and hence specificity. Accounting for predicted binding mode significantly improves prediction accuracy of predicted motifs. This new insight into the fundamental behaviour of C₂H₂-ZFs has implications for both improving the prediction of natural zinc finger-binding sites, and for prioritizing further experiments to complete the code. It also provides a new design feature for zinc finger engineering.     

Synthesis and biological evaluation of 2-substituted-4-(3′,4′,5′-trimethoxyphenyl)-5-aryl thiazoles as anticancer agents

Antitumor agents that bind to tubulin and disrupt microtubule dynamics have attracted considerable attention in the last few years. To extend our knowledge of the thiazole ring as a suitable mimic for the cis-olefin present in combretastatin A-4, we fixed the 3,4,5-trimethoxyphenyl at the C4-position of the thiazole core. We found that the substituents at the C2- and C5-positions had a profound effect on antiproliferative activity. Comparing compounds with the same substituents at the C5-position of the thiazole ring, the moiety at the C2-position influenced antiproliferative activities, with the order of potency being NHCH₃ > Me ? N(CH₃)₂. The N-methylamino substituent significantly improved antiproliferative activity on MCF-7 cells with respect to C2-amino counterparts. Increasing steric bulk at the C2-position from N-methylamino to N,N-dimethylamino caused a 1–2 log decrease in activity. The 2-N-methylamino thiazole derivatives 3b, 3d and 3e were the most active compounds as antiproliferative agents, with IC₅₀ values from low micromolar to single digit nanomolar, and, in addition, they are also active on multidrug-resistant cell lines over-expressing P-glycoprotein. Antiproliferative activity was probably caused by the compounds binding to the colchicines site of tubulin polymerization and disrupting microtubule dynamics. Moreover, the most active compound 3e induced apoptosis through the activation of caspase-2, -3 and -8, but 3e did not cause mitochondrial depolarization.     

Activation of PKR causes amyloid ß-peptide accumulation via de-repression of BACE1 expression

BACE1 is a key enzyme involved in the production of amyloid ß-peptide (Aß) in Alzheimer''s disease (AD) brains. Normally, its expression is constitutively inhibited due to the presence of the 5′untranslated region (5′UTR) in the BACE1 promoter. BACE1 expression is activated by phosphorylation of the eukaryotic initiation factor (eIF)2-alpha, which reverses the inhibitory effect exerted by BACE1 5′UTR. There are four kinases associated with different types of stress that could phosphorylate eIF2-alpha. Here we focus on the double-stranded (ds) RNA-activated protein kinase (PKR). PKR is activated during viral infection, including that of herpes simplex virus type 1 (HSV1), a virus suggested to be implicated in the development of AD, acting when present in brains of carriers of the type 4 allele of the apolipoprotein E gene. HSV1 is a dsDNA virus but it has genes on both strands of the genome, and from these genes complementary RNA molecules are transcribed. These could activate BACE1 expression by the PKR pathway. Here we demonstrate in HSV1-infected neuroblastoma cells, and in peripheral nervous tissue from HSV1-infected mice, that HSV1 activates PKR. Cloning BACE1 5′UTR upstream of a luciferase (luc) gene confirmed its inhibitory effect, which can be prevented by salubrinal, an inhibitor of the eIF2-alpha phosphatase PP1c. Treatment with the dsRNA analog poly (I∶C) mimicked the stimulatory effect exerted by salubrinal over BACE1 translation in the 5′UTR-luc construct and increased Aß production in HEK-APPsw cells. Summarizing, our data suggest that PKR activated in brain by HSV1 could play an important role in the development of AD.