Changes in gene expression in Arabidopsis shoots during phosphate starvation and the potential for developing smart plants

Our aim was to generate and prove the concept of "smart" plants to monitor plant phosphorus (P) status in Arabidopsis. Smart plants can be genetically engineered by transformation with a construct containing the promoter of a gene up-regulated specifically by P starvation in an accessible tissue upstream of a marker gene such as beta-glucuronidase (GUS). First, using microarrays, we identified genes whose expression changed more than 2.5-fold in shoots of plants growing hydroponically when P, but not N or K, was withheld from the nutrient solution. The transient changes in gene expression occurring immediately (4 h) after P withdrawal were highly variable, and many nonspecific, shock-induced genes were up-regulated during this period. However, two common putative cis-regulatory elements (a PHO-like element and a TATA box-like element) were present significantly more often in the promoters of genes whose expression increased 4 h after the withdrawal of P compared with their general occurrence in the promoters of all genes represented on the microarray. Surprisingly, the expression of only four genes differed between shoots of P-starved and -replete plants 28 h after P was withdrawn. This lull in differential gene expression preceded the differential expression of a new group of 61 genes 100 h after withdrawing P. A literature survey indicated that the expression of many of these "late" genes responded specifically to P starvation. Shoots had reduced P after 100 h, but growth was unaffected. The expression of SQD1, a gene involved in the synthesis of sulfolipids, responded specifically to P starvation and was increased 100 h after withdrawing P. Leaves of Arabidopsis bearing a SQD1::GUS construct showed increased GUS activity after P withdrawal, which was detectable before P starvation limited growth. Hence, smart plants can monitor plant P status. Transferring this technology to crops would allow precision management of P fertilization, thereby maintaining yields while reducing costs, conserving natural resources, and preventing pollution.     

Involvement of rho and rho-associated kinase in sphincteric smooth muscle contraction by angiotensin II

The tonic smooth muscles of lower esophageal sphincter (LES) and internal anal sphincter (IAS) are subject to modulation by the neurohumoral agents. We report that angiotensin (Ang) II-induced contraction of rat IAS and LES smooth muscle cells (SMC) was inhibited by Clostridium botulinum C3 exozyme, HA 1077 and Y 27632, suggesting a role for Rho kinase and a Rho-associated kinase (ROK). Ang II-induced contraction of the SMC was also attenuated by genistein, antibodies to the pp60(c-src), p(190) RhoGTPase-activating protein (p190 RhoGAP), carboxyl terminus of Galpha13, carboxyl terminus peptide, and ADP ribosylation factor (ARF) antibody. Ang II-induced increase in p(190) RhoGAP tyrosine phosphorylation was attenuated by genistein. Furthermore, Ang II-induced increase in smooth muscle tone and phosphorylation of myosin light chain (MLC; 20 kDa; MLC20-P) were attenuated by Y 27632 and genistein. The results suggest an important role for Galpha13 and pp60(c-src) in the intracellular events responsible for the activation of RhoA/ROK in Ang II-induced contraction of LES and IAS SMC.     

The translation start codon region is sensitive to antisense PNA inhibition in Escherichia coli

Antisense peptide nucleic acids (PNA) can inhibit bacterial gene expression with gene and sequence specificity. Using attached carrier peptides that aid cell permeation, the antisense effects when targeting essential genes are sufficient to prevent growth and even kill bacteria. However, many design uncertainties remain, including the difficult question of target sequence selection. In this study, we synthesized 90 antisense peptide-PNAs to target sequences in a head to tail manner across the entire length of the mRNA encoding beta-lactamase. The results from this scan pointed to the start codon region as most sensitive to inhibition. To confirm and refine the result, a higher-resolution scan was conducted over the start codon region of the beta-lactamase gene and the essential Escherichia coli acpP gene. For both genes, the start codon region, including the Shine-Dalgarno motif, was sensitive, whereas antisense agents targeted outside of this region were largely ineffective. These results are in accord with natural antisense mechanisms, which typically hinder the start codon region, and the sensitivity of this region should hold true for most bacterial genes as well as for other RNase H-independent antisense agents that rely on a steric blocking mechanism. Therefore, although other design parameters are also important, the start codon region in E. coli mRNA is the most reliable target site for antisense PNAs.     

Liquid chromatography-tandem mass spectrometric quantitative determination of the HIV protease inhibitor atazanavir (BMS-232632) in human peripheral blood mononuclear cells (PBMC): practical approaches to PBMC preparation and PBMC assay design for high-throughput analysis

A selective, accurate, and reproducible LC/MS/MS assay was developed and validated for the determination of the HIV protease inhibitor atazanavir (BMS-232632) in human peripheral blood mononuclear cells (PBMC) samples. In addition to the details of the validated LC/MS/MS method, a practical procedure is described in great detail for the preparation of large supplies of control (blank) PBMC from units of blood (each unit of blood is about 500 ml) for making the calibration standards and quality control (QC) samples. The PBMC assay design, intended for high-throughput sample analysis, is also described in some detail in regards to the composition and concentration expressions of the calibration standards and QC samples, the lysing procedure of the PBMC samples, and the final analysis/quantitation procedure. The method involved automated solid-phase extraction (SPE) of atazanavir and a stable isotope analog internal standard (I.S.) using 3M Empore C2-SD 96-well plates. A portion of the reconstituted sample residue was injected onto a YMC Basic analytical column which was connected to a triple quad mass spectrometer for analyte determination by positive-ion electrospray in the selected reaction monitoring (SRM) mode. The standard curve, which ranged from 5 to 2500 fmol per one million cells (fmol/10(6) cells), was fitted to a quadratic regression model weighted by 1/concentration. The lower limit of quantitation (LLOQ) was 5 fmol/10(6) cells. The inter- and intra-run coefficients of variation (CV) for the assay were <9% and the accuracy was 94-104%. Atazanavir was stable in PBMC for at least 24h at room temperature and for at least 129 days at -15 degrees C.     

Distribution of activated calmodulin in the chloronema tip cells of the moss Funaria hygrometrica

Auxin (indole-3-acetic acid) regulates caulonema differentiation as a result of gradual transitional events in the chloronema tip cells in moss protonema. This auxin action in the moss Funaria hygrometrica involves a rapid influx of calcium ions from the extracellular medium. This investigation demonstrates spatial and temporal changes in calmodulin (CaM) activation (formation of Ca(2+)-CaM complex) in the chloronema tip cells subjected to auxin treatment. Photomicroscopic localisation of the fluorescence (excitation at 365 nm and emission of 397 nm) from the tricomplex of Ca(2+)-CaM with trifluoperazine (TFP, a blocker of Ca(2+)-CaM action) shows a tip to base (tip high) gradient of Ca(2+)-CaM in the chloronema tip cells. Comparison of Ca(2+)-CaM-TFP fluorescence over time in the chloronema tip cells of wild type Funaria with the response in an auxin overproducer mutant (86.1) and an auxin deficient mutant (87.13) reveals the involvement of auxin in calmodulin activation as a rapid response prior to cell differentiation.     

Design and implementation of a domain specific language for phylogenetic inference

Domain experts think and reason at a high level of abstraction when they solve problems in their domain of expertise. We present the design and motivation behind a domain specific language, called phi LOG, to enable biologists to program solutions to phylogenetic inference problems at a very high level of abstraction. The implementation infrastructure (interpreter, compiler, debugger) for the DSL is automatically obtained through a software engineering framework based on Denotational Semantics and Logic Programming.