The TOC159 null mutant of Arabidopsis thaliana is impaired in the accumulation of plastid lipids and phosphatidylcholine.

We used electrospray ionization tandem mass spectrometry to profile glycerolipids in the TOC159 null mutant of Arabidopsis, which is referred to as plastid protein import 2, or ppi2. The goal was to evaluate the impact of a defective atToc159 receptor in the accumulation of plastid lipids. The ppi2 mutant is severely impaired in the accumulation of monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG) and phosphatidylglycerol (PG), which are major components of the thylakoid membranes. Major molecular species of MGDG and DGDG are drastically decreased, which is consistent with our previous findings of decreased levels of hexadecatrienoic and linolenic acids. Under normal growth conditions, the ppi2 mutant accumulated significantly lower levels of phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylinositol (PI). In the cold-acclimated mutant, the amounts of PE and PI were similar to the wildtype level, which indicates that the ER pathway of lipid synthesis was functional in the mutant. The cold-acclimated ppi2 mutant accumulated increased amounts of phosphatidic acid (PA), which was mirrored by an increase in phospholipase Dα (PLDα) transcript levels. These data suggest that PLDα activity contributed to the accumulation of cold-induced PA in the ppi2 mutant. The accumulation of major molecular species in PA indicates that cold-induced PA originated from the degradation of both plastidial and extraplastidial lipids. Compared with the wildtype, the ppi2 mutant had a low double bond index and high acyl chain length, which is indicative of decreased membrane fluidity. Taken together, these data indicate that a defective atToc159 receptor severely impaired the plastid pathway of lipid synthesis, which negatively affected the synthesis and/or accumulation of PC.

A simple method of drying polyacrylamide slab gels that eliminates cracking.

The polyacrylamide slab gel is the most common gel format for analyzing protein samples by electrophoresis. Drying these gels is useful in many biological applications; for example, autoradiography, in which radiolabeled proteins are separated to enable their detection and identification. Dried protein gels can also serve as an ideal method of preserving the gel itself for permanent record-keeping and allowing densitometry at a convenient time. Here I describe a simple and highly reproducible gel-drying method that results in dried gels without the cracks that are frequently encountered with many existing gel-drying methods.

Optimization of western blotting for the detection of proteins of different molecular weight.

Protein samples electroblotted onto nitrocellulose membranes and quenched with a mixture of blocking agents produced a strong signal for cystic fibrosis transmembrane-conductance regulator (CFTR), a high-molecular-weight protein, in western blotting. Optimized conditions for CFTR were then extended to medium- and low-molecular-weight proteins (LAMP1 and Rab11a, respectively) to determine the effects of methanol concentration (0-20%) in Towbin's transfer buffer (TTB). Methanol in TTB appears to have little to no effect on CFTR signal. However, for medium-sized (LAMP1) and small (Rab11a) proteins, a lower concentration of methanol (10%) was sufficient to produce a maximal signal. Therefore, methanol, a toxic solvent, can be removed from or reduced in TTB without compromising signal strength. Here, we show modifications that may be useful in detecting and/or improving the signal of low-abundance proteins.

Effects of Reusing Gel Electrophoresis and Electrotransfer Buffers on Western Blotting.

SDS-PAGE and Western blotting are 2 of the most commonly used biochemical methods for protein analysis. Proteins are electrophoretically separated based on their MWs by SDS-PAGE and then electrotransferred to a solid membrane surface for subsequent protein-specific analysis by immunoblotting, a procedure commonly known as Western blotting. Both of these procedures use a salt-based buffer, with the latter procedure consisting of methanol as an additive known for its toxicity. Previous reports present a contradictory view in favor or against reusing electrotransfer buffer, also known as Towbin's transfer buffer (TTB), with an aim to reduce the toxic waste. In this report, we present a detailed analysis of not only reusing TTB but also gel electrophoresis buffer (EB) on proteins of low to high MW range. Our results suggest that EB can be reused for at least 5 times without compromising the electrophoretic separation of mixture of proteins in an MW standard, BSA, and crude cell lysates. Additionally, reuse of EB did not affect the quality of subsequent Western blots. Successive reuse of TTB, on the other hand, diminished the signal of proteins of different MWs in a protein standard and a high MW membrane protein cystic fibrosis transmembrane-conductance regulator (CFTR) in Western blotting.

A modified protein assay from microgram to low nanogram levels in dilute samples.

In this article, we present a modified and improved protein assay that was previously described as "amidoschwarz assay" by Schaffner and Weissmann. Our improved protein assay is user-friendly and 30-40 times more sensitive than the earlier method. The assay was developed into three formats (macro-, micro-, and nanoassay) with trichloroacetic acid (TCA) as protein precipitating agent, measuring up to 96 samples. The macro and micro formats of this assay require a single reagent staining with amido black of protein dots bound to nitrocellulose membrane with lowest protein measurements to 1 and 0.1 µg, respectively. On the other hand, the nanoassay, with combination staining of amido black followed by colloidal gold, can extend the detection limit to 2.5 ng of protein. Protein concentrations were determined by densitometry and/or spectrophotometry. This assay is compatible with many ionic and non-ionic detergents. This improved protein assay provides an additional choice to researchers in measuring total protein concentration accurately in dilute biological samples as low as 0.125 µg/ml prior to their biochemical analysis such as in comparative proteomics.

Role of tissue-type plasminogen activator in salicylic acid-induced sloughing of human corn tissue.

BACKGROUND: Plasminogen activators (PAs) and their regulatory counterparts, PA inhibitors (PAIs), play a role in normal differentiation processes and various pathophysiologic conditions of the epidermis. Normal desquamation of corneocytes from the skin3s surface may, in part, be regulated by the balanced activities of tissue-type PA (tPA) and PAI-2. Salicylic acid (SA) is commonly used to remove the hyperkeratotic tissue of corns, calluses, and verrucae, and it may disrupt intercellular adhesion structures; however, its exact mechanism of keratolytic action is poorly defined. We sought to determine the effects of SA by comparing the levels of PA and PAI messenger RNA (mRNA) in normal skin, untreated corns, and SA-treated corns. METHODS: Untreated and SA-treated human corn tissue samples were obtained from patients electing surgery to repair bony defects that underlay their lesions. Histopathologic examination of corns was performed by staining the tissue sections with hematoxylin and eosin and by light microscopy. Polymerase chain reaction was used to compare mRNA expression of PAs and PAIs in normal skin, untreated corns, and SA-treated corns. RESULTS: We demonstrated lower tPA and higher PAI-2 mRNA levels in corn tissue compared with normal skin. In corn tissue treated with SA, the expression of tPA mRNA increased and of PAI-2 mRNA decreased to the levels found in normal skin. CONCLUSION: An altered balance in tPA and PAI-2 levels contributes to the induction of hyperkeratotic corn tissue and suggests that the keratolytic action of SA is associated with its ability to stimulate proteinase-meditated desquamation processes.

Methods for RNA extraction, cDNA preparation and analysis of CFTR transcripts.

The scope of this article is to outline some of the basic methods for good quality RNA preparation from mammalian tissues and cells (including epithelial cells). Additionally, we give an outline of common techniques of measuring CFTR gene expression such as quantitative and semi-quantitative reverse transcription (RT) PCR and ribonuclease protection assay (RPA). These methods are designed to detect low abundance transcripts, which apply to CFTR mRNA in most cell types and tissues.

Non-PCR methods for the analysis of CFTR transcripts.

Cystic fibrosis transmembrane conductance regulator gene (CFTR) shows a complex mechanism of tissue-specific and temporal regulation. CFTR mRNA detection and measurement are extremely difficult because of the low to very low levels of its endogenous expression. In this paper, we describe four different non-PCR methods optimized to analyze CFTR transcripts in epithelial cell lines, primary cell lines and native tissues that express significant amounts of CFTR transcript.

Antibodies for CFTR studies.

For most expression studies focusing on the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein, sensitive and specific antibodies (Abs) are critically needed. Several Abs have been produced commercially or by research laboratories for CFTR detection in both cell lines with heterologous or endogenous expression and native cells/tissues. Here, we review the applicability of most Abs currently in use in CF research for the biochemical and/or immunocytochemical detection of CFTR.

Biochemical methods to assess CFTR expression and membrane localization.

Detection of cystic fibrosis transmembrane conductance regulator (CFTR) protein is usually a difficult task to accomplish due to the low levels of expression and high turnover that this membrane protein is submitted to in the cell. Common biochemical methods can be used for the detection of CFTR but several critical points must be taken into account. The scope of this article is to outline biochemical methods commonly used to assess CFTR expression, processing and membrane localization.

Proteomics techniques for cystic fibrosis research.

Numerous factors, other than mutations in the CFTR gene, affect the phenotypic variability of cystic fibrosis (CF). With a two-dimensional electrophoresis (2-DE) analysis of total protein expression profiles (proteomics) of CF versus non-CF cells it is possible to obtain an integrative picture of CF cellular alterations. Through this approach, proteins that interact differently with wild type- and mutant-CFTR can also be identified (interactomics). This can provide insight into CF pathophysiology as well as clues for novel therapeutic targets. Additionally, protein profiling can ultimately identify novel disease markers with the potential for a CF diagnosis not based on the analysis of CFTR gene.

Determination of CFTR chloride channel activity and pharmacology using radiotracer flux methods.

Flux studies using either radioisotopes or ion-selective electrodes are a convenient method to assay the function of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel. Here, we described three different protocols to study the properties, regulation and pharmacology of the CFTR Cl- channel in populations of cells and artificial vesicles. These techniques are widely used to evaluate the function of wild-type and mutant CFTR prior to detailed analyses using the patch-clamp technique. Moreover, they have proved especially valuable in the search for new drugs to treat cystic fibrosis.

A macromolecular complex of beta 2 adrenergic receptor, CFTR, and ezrin/radixin/moesin-binding phosphoprotein 50 is regulated by PKA.

It has been demonstrated previously that both the cystic fibrosis transmembrane conductance regulator (CFTR) and beta(2) adrenergic receptor (beta(2)AR) can bind ezrinradixinmoesin-binding phosphoprotein 50 (EBP50, also referred to as NHERF) through their PDZ motifs. Here, we show that beta(2) is the major adrenergic receptor isoform expressed in airway epithelia and that it colocalizes with CFTR at the apical membrane. beta(2)AR stimulation increases CFTR activity, in airway epithelial cells, that is glybenclamide sensitive. Deletion of the PDZ motif from CFTR uncouples the channel from the receptor both physically and functionally. This uncoupling is specific to the beta(2)AR receptor and does not affect CFTR coupling to other receptors (e.g., adenosine receptor pathway). Biochemical studies demonstrate the existence of a macromolecular complex involving CFTR-EBP50-beta(2)AR through PDZ-based interactions. Assembly of the complex is regulated by PKA-dependent phosphorylation. Deleting the regulatory domain of CFTR abolishes PKA regulation of complex assembly. This report summarizes a macromolecular signaling complex involving CFTR, the implications of which may be relevant to CFTR-dysfunction diseases.