A standardized method for determining buffering capacity of plant foliage

Summary A method for determining the buffering capacity (B.C.) of foliage extracts was standardized and evaluated. Sources of variations (biological, field and laboratory) were identified. These variations were reflected in inter-specific differences, seasonal fluctuations, age of the foliage and duration and the conditions of storage of the extracts. Procedures have been recommended to eliminate or minimize sources of variations (other than inherent specific) by standardizing the field sampling, laboratory processing and methods, and calculations of the buffering capacity. Plants such as lichens, known to be sensitive to air pollutants, had very low B.C. whereas species of intermediate sensitivity such as balsam fir had higher B.C. The B.C. being inherited and significantly different among species, has potential for its use in indexing the relative sensitivity of species to air pollutants especially in areas where large numbers of species are to be compared.     

KIF14 negatively regulates Rap1a–Radil signaling during breast cancer progression

The small GTPase Rap1 regulates inside-out integrin activation and thereby influences cell adhesion, migration, and polarity. Several Rap1 effectors have been described to mediate the cellular effects of Rap1 in a context-dependent manner. Radil is emerging as an important Rap effector implicated in cell spreading and migration, but the molecular mechanisms underlying its functions are unclear. We report here that the kinesin KIF14 associates with the PDZ domain of Radil and negatively regulates Rap1-mediated inside-out integrin activation by tethering Radil on microtubules. The depletion of KIF14 led to increased cell spreading, altered focal adhesion dynamics, and inhibition of cell migration and invasion. We also show that Radil is important for breast cancer cell proliferation and for metastasis in mice. Our findings provide evidence that the concurrent up-regulation of Rap1 activity and increased KIF14 levels in several cancers is needed to reach optimal levels of Rap1–Radil signaling, integrin activation, and cell–matrix adhesiveness required for tumor progression.     

δ-Tocopherol Reduces Lipid Accumulation in Niemann-Pick Type C1 and Wolman Cholesterol Storage Disorders

Niemann-Pick disease type C (NPC) and Wolman disease are two members of a family of storage disorders caused by mutations of genes encoding lysosomal proteins. Deficiency in function of either the NPC1 or NPC2 protein in NPC disease or lysosomal acid lipase in Wolman disease results in defective cellular cholesterol trafficking. Lysosomal accumulation of cholesterol and enlarged lysosomes are shared phenotypic characteristics of both NPC and Wolman cells. Utilizing a phenotypic screen of an approved drug collection, we found that δ-tocopherol effectively reduced lysosomal cholesterol accumulation, decreased lysosomal volume, increased cholesterol efflux, and alleviated pathological phenotypes in both NPC1 and Wolman fibroblasts. Reduction of these abnormalities may be mediated by a δ-tocopherol-induced intracellular Ca²⁺ response and subsequent enhancement of lysosomal exocytosis. Consistent with a general mechanism for reduction of lysosomal lipid accumulation, we also found that δ-tocopherol reduces pathological phenotypes in patient fibroblasts from other lysosomal storage diseases, including NPC2, Batten (ceroid lipofuscinosis, neuronal 2, CLN2), Fabry, Farber, Niemann-Pick disease type A, Sanfilippo type B (mucopolysaccharidosis type IIIB, MPSIIIB), and Tay-Sachs. Our data suggest that regulated exocytosis may represent a potential therapeutic target for reduction of lysosomal storage in this class of diseases.     

Comprehensive and quantitative mapping of energy landscapes for protein-protein interactions by rapid combinatorial scanning

A novel, quantitative saturation (QS) scanning strategy was developed to obtain a comprehensive data base of the structural and functional effects of all possible mutations across a large protein-protein interface. The QS scan approach was applied to the high affinity site of human growth hormone (hGH) for binding to its receptor (hGHR). Although the published structure-function data base describing this system is probably the most extensive for any large protein-protein interface, it is nonetheless too sparse to accurately describe the nature of the energetics governing the interaction. Our comprehensive data base affords a complete view of the binding site and provides important new insights into the general principles underlying protein-protein interactions. The hGH binding interface is highly adaptable to mutations, but the nature of the tolerated mutations challenges generally accepted views about the evolutionary and biophysical pressures governing protein-protein interactions. Many substitutions that would be considered chemically conservative are not tolerated, while conversely, many non-conservative substitutions can be accommodated. Furthermore, conservation across species is a poor predictor of the chemical character of tolerated substitutions across the interface. Numerous deviations from generally accepted expectations indicate that mutational tolerance is highly context dependent and, furthermore, cannot be predicted by our current knowledge base. The type of data produced by the comprehensive QS scan can fill the gaps in the structure-function matrix. The compilation of analogous data bases from studies of other protein-protein interactions should greatly aid the development of computational methods for explaining and designing molecular recognition.     

Comparative structural analysis of the Erbin PDZ domain and the first PDZ domain of ZO-1. Insights into determinants of PDZ domain specificity

We report a structural comparison of the first PDZ domain of ZO-1 (ZO1-PDZ1) and the PDZ domain of Erbin (Erbin-PDZ). Although the binding profile of Erbin-PDZ is extremely specific ([D/E][T/S]WV(COOH)), that of ZO1-PDZ1 is similar ([R/K/S/T][T/S][W/Y][V/I/L](COOH)) but broadened by increased promiscuity for three of the last four ligand residues. Consequently, the biological function of ZO-1 is also broadened, as it interacts with both tight and adherens junction proteins, whereas Erbin is restricted to adherens junctions. Structural analyses reveal that the differences in specificity can be accounted for by two key differences in primary sequence. A reduction in the size of the hydrophobic residue at the base of the site(0) pocket enables ZO1-PDZ1 to accommodate larger C-terminal residues. A single additional difference alters the specificity of both site(-1) and site(-3). In ZO1-PDZ1, an Asp residue makes favorable interactions with both Tyr(-1) and Lys/Arg(-3). In contrast, Erbin-PDZ contains an Arg at the equivalent position, and this side chain cannot accommodate either Tyr(-1) or Lys/Arg(-3) but, instead, interacts favorably with Glu/Asp(-3). We propose a model for ligand recognition that accounts for interactions extending across the entire binding site but that highlights several key specificity switches within the PDZ domain fold.     

Dopamine D1 receptor-mediated toxicity in human SK-N-MC neuroblastoma cells

Striatal degeneration occurs through unknown mechanisms in certain neurodegenerative disorders characterized by increased and sustained synaptic levels of dopamine. In the present studies, we examined the effects of treatment of SK-N-MC neuroblastoma cells with dopamine to understand the participation of dopamine D(1) receptor in postsynaptic cytotoxicity. Treatment of SK-N-MC cells either with dopamine or the D(1) receptor agonist SKF R-38393 resulted in a significant increase in the production of reactive oxygen species (by approximately 2.75-fold) and cell death ( approximately 50%), while antagonism of the D(1) receptor with SCH 23390 significantly reversed (to approximately 75% of control level) these effects. Accumulation of cAMP in dopamine treated cells (t(1/2)=1.5h) preceded changes in ionic gradient (t(1/2)=6.5h), as measured by intracellular potassium concentration and leakage of cytochrome c into the cytosol (t(1/2)=13 h), suggesting a possible staging of toxic events as a result of activation of D(1) receptor by dopamine. Examination of cellular metabolic properties with (13)C NMR spectroscopy showed an inhibitory effect on tricarboxylic acid cycle metabolism via D(1)-mediated receptors after treatment with dopamine, suggesting a direct role for D(1) receptor in dopamine-induced postsynaptic cell death. The present studies provide novel insight into a possible patho-physiological staging of cytotoxic events that are mediated by activation of D(1) receptor.     

Regeneration of fertile green plants from oat isolated microspore culture

Regeneration of fertile green plants from isolated oat microspores is reported for the first time. Factors critical for microspore growth and regeneration include cold pre-treatment, pH of culture medium and the use of conditioned culture medium. It was found that cold pre-treatment at 4°C in the dark for a minimum of 6 weeks was necessary to consistently achieve microspore growth into multicellular structures (MCS). Longer pre-treatments of up to 9 weeks were tested and found to be positively correlated with the number of MCS produced. Microspore culture medium with pH 8.0 produced significantly more MCS larger than eight cells in size than media with pH 5.8. The use of medium conditioned by actively growing barley microspores significantly increased the numbers of MCS larger than eight cells in size compared to non-conditioned media. Plants were regenerated only from cultures using conditioned medium. A total of 2 green plants and 15 albinos were regenerated. Of the green plants, one had the haploid chromosome complement (n = 3x = 21) and the other had the parental hexaploid chromosome complement (2n = 6x = 42) which may be due to spontaneous chromosome doubling. The hexaploid plant set seed naturally and the haploid plant set seed after its chromosome complement was doubled with colchicine.     

Use of high-density tiling microarrays to identify mutations globally and elucidate mechanisms of drug resistance in Plasmodium falciparum

Background

The identification of genetic changes that confer drug resistance or other phenotypic changes in pathogens can help optimize treatment strategies, support the development of new therapeutic agents, and provide information about the likely function of genes. Elucidating mechanisms of phenotypic drug resistance can also assist in identifying the mode of action of uncharacterized but potent antimalarial compounds identified in high-throughput chemical screening campaigns against Plasmodium falciparum.

Results

Here we show that tiling microarrays can detect de novo a large proportion of the genetic changes that differentiate one genome from another. We show that we detect most single nucleotide polymorphisms or small insertion deletion events and all known copy number variations that distinguish three laboratory isolates using readily accessible methods. We used the approach to discover mutations that occur during the selection process after transfection. We also elucidated a mechanism by which parasites acquire resistance to the antimalarial fosmidomycin, which targets the parasite isoprenoid synthesis pathway. Our microarray-based approach allowed us to attribute in vitro derived fosmidomycin resistance to a copy number variation event in the pfdxr gene, which enables the parasite to overcome fosmidomycin-mediated inhibition of isoprenoid biosynthesis.

Conclusions

We show that newly emerged single nucleotide polymorphisms can readily be detected and that malaria parasites can rapidly acquire gene amplifications in response to in vitro drug pressure. The ability to define comprehensively genetic variability in P. falciparum with a single overnight hybridization creates new opportunities to study parasite evolution and improve the treatment and control of malaria.     

MOTIPS: Automated Motif Analysis for Predicting Targets of Modular Protein Domains

Background  

Many protein interactions, especially those involved in signaling, involve short linear motifs consisting of 5-10 amino acid residues that interact with modular protein domains such as the SH3 binding domains and the kinase catalytic domains. One straightforward way of identifying these interactions is by scanning for matches to the motif against all the sequences in a target proteome. However, predicting domain targets by motif sequence alone without considering other genomic and structural information has been shown to be lacking in accuracy.