Construction and Characterization of a Magnaporthe grisea Bacterial Artificial Chromosome Library

Diaz-Perez, S. V., Crouch, V. W., and Orbach, M. J. 1996. Construction and characterization of a Magnaporthe grisea bacterial artificial chromosome library. Fungal Genet. Biol. 20, 280-288. A bacterial artificial chromosome (BAC) library of Magnaporthe grisea containing 4128 clones with an average insert size of 66-kb has been constructed. This library represents seven genome equivalents of M. grisea and has been demonstrated to be representative of the genome by screening for the presence of several single-copy genes and DNA markers. The utility of the library for use in map-based cloning projects was shown by the spanning of a nine-cosmid, 207-kb DNA contig with only 3 BAC clones. In addition, using a lys1-3 auxotroph, we have shown that BAC clones at least 113 kb can be transformed into M. grisea to screen for complementation of mutations. Thus, BACs isolated in chromosome walks can be rapidly screened for the presence of the sought after gene. The ease of construction of BAC libraries and of isolation and manipulation of BAC clones makes the BAC system an ideal one for physical analyses of fungal genomes.     

In vivo and in vitro processing of seed reserve protein in the endoplasmic reticulum: evidence for two glycosylation steps

Cotyledons of the common bean (Phaseolus vulgaris L.) synthesize large amounts of the reserve protein phaseolin. The polypeptides are synthesized on membrane-bound polysomes, pass through the endoplasmic reticulum (ER) and accumulate in protein bodies. For a study of the biosynthesis and processing of phaseolin, developing cotyledons were labeled with radioactive amino acids, glucosamine and mannose, and isolated fractions (polysomal RNA, polysomes, and rough ER) were used for in vitro protein synthesis. Newly synthesized phaseolin present in the ER of developing cotyledons can be fractioned into four glycopolypeptides by SDS PAGE. In vitro synthesis with polysomal RNA results in the formation of two polypeptides by polysome run-off shows that glycosylation is a co-translational event. The two unglycosylated polypeptides formed by polysome run-off are slightly smaller than the two polypeptides formed by in vitro translation of isolated RNA, indicating that a signal peptide may be present on these polypeptides. Run-off synthesis with rough ER produces a pattern of four polypeptides similar to the one obtained by in vivo labeling. The two abundant glycopolypeptides formed by polysome run-off. This result indicates the existence of a second glycosylation event for the abundant polypeptides. Inhibition of glycosylation by Triton X-100 during chain-completion with rough ER was used to show that these two glycosylation steps normally occur sequentially. Both glycosylation steps are inhibited by tunicamycin. Analysis of carhohydrate to protein ratios of the different polypeptides and of trypsin digests of polypeptides labeled with [(3)H]glucosamine confirmed the conclusion that some glycosylated polypeptides contain two oligosaccharide chains, while others contain only one. An analysis of tryptic peptide maps shows that each of the unglycosylated polypeptides is the precursor for one glycosylated polypeptide with one oligosaccharide chain and one with two oligosaccharide chains.     

Cultured endothelial cells produce heparinlike inhibitor of smooth muscle cell growth

Using cultured cells from bovine and rat aortas, we have examined the possibility that endothelial cells might regulate the growth of vascular smooth muscle cells. Conditioned medium from confluent bovine aortic endothelial cells inhibited the proliferation of growth-arrested smooth muscle cells. Conditioned medium from exponential endothelial cells, and from exponential or confluent smooth muscle cells and fibroblasts, did not inhibit smooth muscle cell growth. Conditioned medium from confluent endothelial cells did not inhibit the growth of endothelial cells or fibroblasts. In addition to the apparent specificity of both the producer and target cell, the inhibitory activity was heat stable and not affected by proteases. It was sensitive flavobacterium heparinase but not to hyaluronidase or chondroitin sulfate ABC lyase. It thus appears to be a heparinlike substance. Two other lines of evidence support this conclusion. First, a crude isolate of glycosaminoglycans (TCA-soluble, ethanol-precipitable material) from endothelial cell-conditioned medium reconstituted in 20 percent serum inhibited smooth muscle cell growth; glycosaminoglycans isolated from unconditioned medium (i.e., 0.4 percent serum) had no effect on smooth muscle cell growth. No inhibition was seen if the glycosaminoglycan preparation was treated with heparinase. Second, exogenous heparin, heparin sulfate, chondroitin sulfate B (dermatan sulfate), chondroitin sulfate ABC, and hyaluronic acid were added to 20 percent serum and tested for their ability to inhibit smooth muscle cell growth. Heparin inhibited growth at concentrations as low as 10 ng/ml. Other glycosaminoglycans had no effect at doses up to 10 μg/ml. Anticoagulant and non- anticoagulant heparin were equally effective at inhibiting smooth muscle cell growth, as they were in vivo following endothelial injury (Clowes and Karnovsk. Nature (Lond.). 265:625-626, 1977; Guyton et al. Circ. Res. 46:625-634, 1980), and in vitro following exposure of smooth muscle cells to platelet extract (Hoover et al. Circ. Res. 47:578-583, 1980). We suggest that vascular endothelial cells may secrete a heparinlike substance in vivo which may regulate the growth of underlying smooth muscle cells.     

Substructure of the glomerular slit diaphragm in freeze-fractured normal rat kidney

In the renal glomerulus, the narrow slits between adjacent epithelial podocytes are bridged by a diaphragm (2, 8, 11). In rat and mouse kidneys fixed by perfusion with tannic acid and glutaraldehyde (TAG), it has recently been discovered that this diaphragm has a highly ordered, isoporous substructure (9). It consists of a regular array of alternating cross bridges extending from the podocyte plasma membranes to a centrally running filament. This zipperlike pattern results in two rows of rectangular pores, approximately 40 X 140 A in cross section, dimensions consistent with the proposed role of the diaphragm as an important filtration barrier to plasma proteins (6). In the present study, we found in freeze-cleaved and in freeze-etched normal rat glomeruli that the surface of the slit diaphragm has an appearance conforming to the pattern found in sectioned material.     

Psychological stress reduces cyclic 3',5'-adenosine monophosphate levels in the cerebral cortex of conscious rats, as determined by a new cryogenic method of rapid tissue fixation.

Abstract— Cryoplates were implanted on the surface of the cortex in 32 chronic rat preparations. These devices were used both to freeze and to extract small samples of tissue. Coolant was circulated through each device by small flexible polyethylene tubes. Two series of experiments were performed. In the first, the animals were unrestrained and showed no behavioral signs of stress during the freeze fixation. The temperature responses of the cryoplates were very rapid (?632°C/s), and samples more than 1 mm thick were frozen and extracted within a few hundred ms following the onset of cooling. Each sample was analyzed for 3′.5′-adenosine monophosphate (cyclic AMP) and protein content. The results from the cryoplate group (25.6 ± 15.6pmol cyclic AMP/mg protein) were compared to those obtained from two other groups in which freeze fixation was produced by immersion in liquid nitrogen (13.6 ± 4.6pmol/mg protein) or decapitation into liquid nitrogen (18.6 + 7.6pmol/mg protein). In the second series of experiments, three types of stress (limb restraint, non-adaptation to the experimental situation, and moderate cutaneous electric shock) were induced separately in order to determine the influence of each on cortical levels of cyclic AMP. Control animals were highly adapted to the experimental situation, freely moving and not shocked. The samples from each of the stressed groups showed a statistically significant (P≤. 0.01) reduction in cyclic AMP in comparison with the level in the controls (control: 29.3 pmol/mg protein; restrained: 14.2pmol/mg protein; unadapted: 9.6pmol/mg protein; shocked: 7.1 pmol/mg protein). Thus, psychological and physical stress reduced cyclic AMP content in parietal cortex. Results from the second series of experiments suggest that the significantly higher mean and larger standard deviation of the cryoplate group in the first series are due to less psychological and physical stress being evoked by our method; different types of stress appear to account for the two different lower levels found in the immersion and decapitation groups. We believe that our method of cryogenic tissue fixation offers an improved approach to study of the neurochemical correlates of behavioral and neuroelectric events in the conscious animal.     

Carcinoembryonic antigen production by human colorectal adenocarcinoma cells in matrix-perfusion culture

Summary Four human colorectal adenocarcinoma tumor cell lines, previously established and characterized in monolayer culture were grown in a matrix-perfusion culture system to determine the suitability of this technique for synthesis of carcinoembryonic antigen (CEA). Production of CEA in excess of 100,000 ng was attained from one cell line, SW 403, during 15-day growth trials. In growth trials and cell-free diffusion studies, CEA passed through membranes of 100,000-dalton molecular weight porosity but not 10,000 porosity. Using cell cultures of high, moderate, or low producers, CEA synthesis tended to reach a plateau after several days of culture and remained nearly constant as the cells attained a maintenance condition. Basic biologic characteristics of the cell lines, expressed as growth rates and CEA produced per 10⁶ cells, were comparable in monolayer and perfusion culture. The high cell densities, (10⁸ to 10⁹ cells per ml) achieved in matrix perfusion made it possible to routinely obtain continuous high yields of CEA over an extended time period. Presented in part at the 29th Annual Meeting of the Tissue Culture Association, Denver, Colorado, June 4–8, 1978. This work was supported in part by the fund for Organized Research, State of Texas, and a grant from Scott and White Clinic.     

Studies on the mechanism for entry of vesicular stomatitis virus glycoprotein g mRNA into membrane-bound polyribosome complexes

Glycoprotein mRNA (G mRNA) of vesicular stomatitis virus is synthesized in the cytosol fraction of infected HeLa cells. Shortly after synthesis, this mRNA associates with 40S ribosomal subunits and subsequently forms 80S monosomes in the cytosol fraction. The bulk of labeled G mRNA is then found in polysomes associated with the membrane, without first appearing in the subunit or monomer pool of the membrane-bound fraction. Inhibition of the initiation of protein synthesis by pactamycin or muconomycin A blocks entry of newly synthesized G m RNA into membrane-bound polysomes. Under these circumstances, labeled G mRNA accumulates into the cytosol. Inhibition of the elongation of protein synthesis by cucloheximide, however, allows entry of 60 percent of newly synthesized G mRNA into membrane-bound polysomes. Furthermore, prelabeled G mRNA associated with membrane-bound polysomes is released from the membrane fraction in vivo by pactamycin or mucomycon A and in vitro by 1mM puromycin - 0.5 M KCI. This release is not due to nonspecific effects of the drugs. These results demonstrate that association of G mRNA with membrane-bound polysomes is dependent upon polysome formation and initiation of protein synthesis. Therefore, direct association of the 3' end of G mRNA with the membrane does not appear to be the initial event in the formation of membrane-bound polysomes.     

Improved Normalization of Systematic Biases Affecting Ion Current Measurements in Label-free Proteomics Data

Normalization is an important step in the analysis of quantitative proteomics data. If this step is ignored, systematic biases can lead to incorrect assumptions about regulation. Most statistical procedures for normalizing proteomics data have been borrowed from genomics where their development has focused on the removal of so-called ‘batch effects.’ In general, a typical normalization step in proteomics works under the assumption that most peptides/proteins do not change; scaling is then used to give a median log-ratio of 0. The focus of this work was to identify other factors, derived from knowledge of the variables in proteomics, which might be used to improve normalization. Here we have examined the multi-laboratory data sets from Phase I of the NCI''s CPTAC program. Surprisingly, the most important bias variables affecting peptide intensities within labs were retention time and charge state. The magnitude of these observations was exaggerated in samples of unequal concentrations or “spike-in” levels, presumably because the average precursor charge for peptides with higher charge state potentials is lower at higher relative sample concentrations. These effects are consistent with reduced protonation during electrospray and demonstrate that the physical properties of the peptides themselves can serve as good reporters of systematic biases. Between labs, retention time, precursor m/z, and peptide length were most commonly the top-ranked bias variables, over the standardly used average intensity (A). A larger set of variables was then used to develop a stepwise normalization procedure. This statistical model was found to perform as well or better on the CPTAC mock biomarker data than other commonly used methods. Furthermore, the method described here does not require a priori knowledge of the systematic biases in a given data set. These improvements can be attributed to the inclusion of variables other than average intensity during normalization.The number of laboratories using MS as a quantitative tool for protein profiling continues to grow, propelling the field forward past simple qualitative measurements (i.e. cataloging), with the aim of establishing itself as a robust method for detecting proteomic differences. By analogy, semiquantitative proteomic profiling by MS can be compared with measurement of relative gene expression by genomics technologies such as microarrays or, newer, RNAseq measurements. While proteomics is disadvantaged by the lack of a molecular amplification system for proteins, successful reports from discovery experiments are numerous in the literature and are increasing with advances in instrument resolution and sensitivity.In general, methods for performing relative quantitation can be broadly divided into two categories: those employing labels (e.g. iTRAQ, TMT, and SILAC (1)) and so-called “label-free” techniques. Labeling methods involve adding some form of isobaric or isotopic label(s) to the proteins or peptides prior to liquid chromatography-tandem MS (LC-MS/MS) analysis. Chemical labels are typically applied during sample processing, and isotopic labels are commonly added during cell culture (i.e. metabolic labeling). One advantage of label-based methods is that the two (or more) differently-labeled samples can be mixed and run in single LC-MS analyses. This is in contrast to label-free methods which require the samples to be run independently and the data aligned post-acquisition.Many labs employ label-free methods because they are applicable to a wider range of samples and require fewer sample processing steps. Moreover, data from qualitative experiments can sometimes be re-analyzed using label-free software tools to provide semiquantitative data. Advances in these software tools have been extensively reviewed (2). While analysis of label-based data primarily uses full MS scan (MS1)¹ or tandem MS scan (MS2) ion current measurements, analysis of label-free data can employ simple counts of confidently identified tandem mass spectra (3). So-called spectral counting makes the assumption that the number of times a peptide is identified is proportional to its concentration. These values are sometimes summed across all peptides for a given protein and scaled by protein length. Relative abundance can then be calculated for any peptide or protein of interest. While this approach may be easy to perform, its usefulness is particularly limited in smaller data sets and/or when counts are low.This report focuses only on the use of ion current measurements in label-free data sets, specifically those calculated from extracted MS1 ion chromatograms (XICs). In general terms, raw intensity values (i.e. ion counts in arbitrary units) cannot be used for quantitation in the absence of cognate internal standards because individual ion intensities depend on a response factor, related to the chemical properties of the molecule. Intensities are instead almost always reserved for relative determinations. Furthermore, retention times are sometimes used to align the chromatograms between runs to ensure higher confidence prior to calculating relative intensities. This step is crucial for methods without corresponding identity information, particularly for experiments performed on low-resolution instruments. To support a label-free workflow, peptide identifications are commonly made from tandem mass spectra (MS/MS) acquired along with direct electrospray signal (MS1). Or, in alternative workflows seeking deeper coverage, interesting MS1 components can be targeted for identification by MS/MS in follow-up runs (4).“Rolling up” the peptide ion information to the peptide and protein level is also done in different ways in different labs. In most cases, “peptide intensity” or “peptide abundance” is the summed or averaged value of the identified peptide ions. How the peptide information is transferred to the protein level differs between methods but typically involves summing one or more peptide intensities, following parsimony analysis. One such solution is the “Top 3” method developed by Silva and co-workers (5).Because peptides in label-free methods lack labeled analogs and require separate runs, they are more susceptible to analytical noise and systematic variations. Sources of these obscuring variations can come from many sources, including sample preparation, operator error, chromatography, electrospray, and even from the data analysis itself. While analytical noise (e.g. chemical interference) is difficult to selectively reject, systematic biases can often be removed by statistical preprocessing. The goal of these procedures is to normalize the data prior to calculations of relative abundance. Failure to resolve these issues is the common origin of batch effects, previously described for genomics data, which can severely limit meaningful interpretation of experimental data (6, 7).These effects have also been recently explored in proteomics data (8). Methods used to normalize proteomics data have been largely borrowed from the microarray community, or are based on a simple mean/median intensity ratio correction. Methods applied on microarray and/or gene chip and used on proteomics data include scaling, linear regression, nonlinear regression, and quantile normalizations (9). Moreover, work has also been done to improve normalization by subselecting a peptide basis (10). Other work suggests that linear regression, followed by run order analysis, works better than other methods tested (11). Key to this last method is the incorporation of a variable other than intensity during normalization. It is also important to note that little work has been done towards identifying the underlying sources of these variations in proteomics data. Although cause-and-effect is often difficult to determine, understanding these relationships will undoubtedly help remove and avoid the major underlying sources of systematic variations.In this report, we have attempted to combine our efforts focused on understanding variability with the work initiated by others for normalizing ion current-based label-free proteomics data. We have identified several major variables commonly affecting peptide ion intensities both within and between labs. As test data, we used a subset of raw data acquired during Phase I of the National Cancer Institute''s (NCI) Clinical Proteomics Technology Assessment for Cancer (CPTAC) program. With these data, we were able to develop a statistical model to rank bias variables and normalize the intensities using stepwise, semiparametric regression. The data analysis methods have been implemented within the National Institute of Standards and Technology (NIST) MS quality control (MSQC) pipeline. Finally, we have developed R code for removing systematic biases and have tested it using a reference standard spiked into a complex biological matrix (i.e. yeast cell lysate).     

Intestinal barrier function in response to abundant or depleted mucosal glutathione in Salmonella-infected rats

Background  

Glutathione, the main antioxidant of intestinal epithelial cells, is suggested to play an important role in gut barrier function and prevention of inflammation-related oxidative damage as induced by acute bacterial infection. Most studies on intestinal glutathione focus on oxidative stress reduction without considering functional disease outcome. Our aim was to determine whether depletion or maintenance of intestinal glutathione changes susceptibility of rats to Salmonella infection and associated inflammation.