Cross talk between energy cost and expression of Methyl Jasmonate-regulated genes: from DNA to protein

Journal of Plant Biochemistry and Biotechnology - Plant cell balances energy consumption for its different biological processes under oxidative circumstances. The aim of the present study is to...     

Cryosectioning Yeast Communities for Examining Fluorescence Patterns

Microbes typically live in communities. The spatial organization of cells within a community is believed to impact the survival and function of the community¹. Optical sectioning techniques, including confocal and two-photon microscopy, have proven useful for observing spatial organization of bacterial and archaeal communities^2,3. A combination of confocal imaging and physical sectioning of yeast colonies has revealed internal organization of cells⁴. However, direct optical sectioning using confocal or two-photon microscopy has been only able to reach a few cell layers deep into yeast colonies. This limitation is likely because of strong scattering of light from yeast cells⁴.Here, we present a method based on fixing and cryosectioning to obtain spatial distribution of fluorescent cells within Saccharomyces cerevisiae communities. We use methanol as the fixative agent to preserve the spatial distribution of cells. Fixed communities are infiltrated with OCT compound, frozen, and cryosectioned in a cryostat. Fluorescence imaging of the sections reveals the internal organization of fluorescent cells within the community.Examples of yeast communities consisting of strains expressing red and green fluorescent proteins demonstrate the potentials of the cryosectioning method to reveal the spatial distribution of fluorescent cells as well as that of gene expression within yeast colonies^2,3. Even though our focus has been on Saccharomyces cerevisiae communities, the same method can potentially be applied to examine other microbial communities.     

Loss of caveolin-1 gene expression accelerates the development of dysplastic mammary lesions in tumor-prone transgenic mice

Caveolin-1 is the principal structural component of caveolae microdomains, which represent a subcompartment of the plasma membrane. Several independent lines of evidence support the notion that caveolin-1 functions as a suppressor of cell transformation. For example, the human CAV-1 gene maps to a suspected tumor suppressor locus (D7S522/7q31.1) that is frequently deleted in a number of carcinomas, including breast cancers. In addition, up to 16% of human breast cancers harbor a dominant-negative mutation, P132L, in the CAV-1 gene. Despite these genetic associations, the tumor suppressor role of caveolin-1 still remains controversial. To directly assess the in vivo transformation suppressor activity of the caveolin-1 gene, we interbred Cav-1 (-/-) null mice with tumor-prone transgenic mice (MMTV-PyMT) that normally develop multifocal dysplastic lesions throughout the entire mammary tree. Herein, we show that loss of caveolin-1 gene expression dramatically accelerates the development of these multifocal dysplastic mammary lesions. At 3 wk of age, loss of caveolin-1 resulted in an approximately twofold increase in the number of lesions (foci per gland; 3.3 +/- 1.0 vs. 7.0 +/- 1.2) and an approximately five- to sixfold increase in the total area occupied by these lesions. Similar results were obtained at 4 wk of age. However, complete loss of caveolin-1 was required to accelerate the appearance of these dysplastic mammary lesions, because Cav-1 (+/-) heterozygous mice did not show any increases in foci development. We also show that loss of caveolin-1 increases the extent and the histological grade of these mammary lesions and facilitates the development of papillary projections in the mammary ducts. Finally, we demonstrate that cyclin D1 expression levels are dramatically elevated in Cav-1 (-/-) null mammary lesions, consistent with the accelerated appearance and growth of these dysplastic foci. This is the first in vivo demonstration that caveolin-1 can function as a transformation suppressor gene.     

Caveolin-1 null (-/-) mice show dramatic reductions in life span

Caveolae are 50-100 nm flask-shaped invaginations of the plasma membrane found in most cell types. Caveolin-1 is the principal protein component of caveolae membranes in nonmuscle cells. The recent development of Cav-1-deficient mice has allowed investigators to study the in vivo functional role of caveolae in the context of a whole animal model, as these mice lack morphologically detectable caveolae membrane domains. Surprisingly, Cav-1 null mice are both viable and fertile. However, it remains unknown whether loss of caveolin-1 significantly affects the overall life span of these animals. To quantitatively determine whether loss of Cav-1 gene expression confers any survival disadvantages with increasing age, we generated a large cohort of mice (n = 180), consisting of Cav-1 wild-type (+/+) (n = 53), Cav-1 heterozygous (+/-) (n = 70), and Cav-1 knockout (-/-) (n = 57) animals, and monitored their long-term survival over a 2 year period. Here, we show that Cav-1 null (-/-) mice exhibit an approximately 50% reduction in life span, with major declines in viability occurring between 27 and 65 weeks of age. However, Cav-1 heterozygous (+/-) mice did not show any changes in long-term survival, indicating that loss of both Cav-1 alleles is required to mediate a reduction in life span. Mechanistically, these dramatic reductions in life span appear to be secondary to a combination of pulmonary fibrosis, pulmonary hypertension, and cardiac hypertrophy in Cav-1 null mice. Taken together, our results provide the first demonstration that loss of Cav-1 gene expression and caveolae organelles dramatically affects the long-term survival of an organism. In addition, aged Cav-1 null mice may provide a new animal model to study the pathogenesis and treatment of progressive hypertrophic cardiomyopathy and sudden cardiac death syndrome.     

RESOURCERER: a database for annotating and linking microarray resources within and across species

Microarray expression analysis is providing unprecedented data on gene expression in humans and mammalian model systems. Although such studies provide a tremendous resource for understanding human disease states, one of the significant challenges is cross-referencing the data derived from different species, across diverse expression analysis platforms, in order to properly derive inferences regarding gene expression and disease state. To address this problem, we have developed RESOURCERER, a microarray-resource annotation and cross-reference database built using the analysis of expressed sequence tags (ESTs) and gene sequences provided by the TIGR Gene Index (TGI) and TIGR Orthologous Gene Alignment (TOGA) databases [now called Eukaryotic Gene Orthologs (EGO)].     

Adenovirus-mediated transmission of a dominant negative transforming growth factor-beta receptor inhibits in vitro mouse cranial suture fusion

Recent studies have implicated the transforming growth factor (TGF)-beta family in the regulation of pathological sporadic cranial suture fusion. In addition, these studies have shown that TGF-beta is highly expressed by the dura mater underlying fusing murine cranial sutures. The purpose of the present experiments was to analyze the effects of disrupting TGF-beta signaling during programmed mouse cranial suture fusion. Using recombinant DNA technology, a replication-deficient adenovirus encoding a defective TGF-beta receptor (Ad.DN-TbetaRII) capable of blocking TGF-beta biological activity was constructed. Mouse posterior frontal sutures were harvested before the initiation of suture fusion (postnatal day 25), and the dura mater underlying the suture was infected with vehicle, Ad.DN-TbetaRII, or control virus (Ad.LacZ; n = 10 each). Sutures were cultured for 14 or 30 days in an organ culture system and analyzed macroscopically and histologically.X-gal staining of Ad.LacZ-infected sutures 14 days after culture revealed strong staining of cells localized to the dura mater. Macroscopic analysis revealed complete sutural fusion in vehicle and Ad.LacZ-infected sutures. In contrast, Ad.DN-TBRII-infected sutures demonstrated nearly complete patency. Histological analysis confirmed our macroscopic observations with sutural fusion in 81.3 +/- 10 percent and 74.5 +/- 9 percent of vehicle and Ad.LacZ-infected sutures, respectively, versus 38.1 +/- 12 percent (p < 0.001) in Ad.DN-TbetaRII-infected sutures. In addition, transfection with the Ad.DN-TbetaRII virus resulted in a significant attenuation of anterior-to-posterior suture fusion, with the majority of fused sections localized to anterior sections. These data strongly implicate TGF-beta biological activity in the dura mater underlying the posterior frontal suture in the regulation of programmed sutural fusion. In addition, this study demonstrates the utility of adenovirus-mediated gene transfer in preventing programmed sutural fusion.     

Elucidating the exact role of engineered CRABPII residues for the formation of a retinal protonated Schiff base

Cellular Retinoic Acid Binding Protein II (CRABPII) has been reengineered to specifically bind and react with all‐trans‐retinal to form a protonated Schiff base. Each step of this process has been dissected and four residues (Lys132, Tyr134, Arg111, and Glu121) within the CRABPII binding site have been identified as crucial for imine formation and/or protonation. The precise role of each residue has been examined through site directed mutagenesis and crystallographic studies. The crystal structure of the R132K:L121E‐CRABPII (PDB‐3I17) double mutant suggests a direct interaction between engineered Glu121 and the native Arg111, which is critical for both Schiff base formation and protonation. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Estimation of anisotropy coefficient of swine pancreas,liver and muscle at 1064 nm based on goniometric technique

Optical properties of tissues are required for theoretical modeling of Laser Ablation in tumor therapy. The light scattering characteristic of tissues is described by the anisotropy coefficient, g. The relationship between the angular distribution of scattered light and g is given by the Henyey‐Greenstein (HG) phase function. This work describes the estimation of anisotropy coefficients of ex vivo swine pancreas, liver and muscle at 1064 nm. The intensities of scattered light at fixed angles were measured under repeatability conditions. Experimental data were fitted with a two‐term HG, estimating the anisotropy coefficients for the forward (e.g., 0.956 for pancreas, 0.964 for liver and 0.968 for muscle) and the backward (e.g., –0.481 for pancreas, –0.414 for liver and –0.372 for muscle) scattering.

Experimental set up employed to estimate the anisotropy coefficient of biological tissues. The image on the left depicts the holder used to house tissue, laser fiber and photodetector; on the left an example of scattered light beam is shown, as well as the effect due to Snell's law.     

Protein secondary structure prediction using NMR chemical shift data

Accurate determination of protein secondary structure from the chemical shift information is a key step for NMR tertiary structure determination. Relatively few work has been done on this subject. There needs to be a systematic investigation of algorithms that are (a) robust for large datasets; (b) easily extendable to (the dynamic) new databases; and (c) approaching to the limit of accuracy. We introduce new approaches using k-nearest neighbor algorithm to do the basic prediction and use the BCJR algorithm to smooth the predictions and combine different predictions from chemical shifts and based on sequence information only. Our new system, SUCCES, improves the accuracy of all existing methods on a large dataset of 805 proteins (at 86% Q(3) accuracy and at 92.6% accuracy when the boundary residues are ignored), and it is easily extendable to any new dataset without requiring any new training. The software is publicly available at http://monod.uwaterloo.ca/nmr/succes.