Crystallographic and mutational studies of Mycobacterium tuberculosis recA mini-inteins suggest a pivotal role for a highly conserved aspartate residue

The 440 amino acid Mtu recA intein consists of independent protein-splicing and endonuclease domains. Previously, removal of the central endonuclease domain of the intein, and selection for function, generated a 168 residue mini-intein, DeltaI-SM, that had splicing activity similar to that of the full-length, wild-type protein. A D422G mutation (DeltaI-CM) increased C-terminal cleavage activity. Using the DeltaI-SM mini-intein structure (presented here) as a guide, we previously generated a highly active 139 residue mini-intein, DeltaDeltaI(hh)-SM, by replacing 36 amino acid residues in the residual endonuclease loop with a seven-residue beta-turn from the autoprocessing domain of Hedgehog protein. The three-dimensional structures of DeltaI-SM, DeltaDeltaI(hh)-SM, and two variants, DeltaDeltaI(hh)-CM and DeltaDeltaI(hh), have been determined to evaluate the effects of the minimization on intein integrity and to investigate the structural and functional consequences of the D422G mutation. These structural studies show that Asp422 is capable of interacting with both the N and C termini. These interactions are lacking in the CM variant, but are replaced by contacts with water molecules. Accordingly, additional mutagenesis of residue 422, combined with mutations that isolate N-terminal and C-terminal cleavage, showed that the side-chain of Asp422 plays a role in both N and C-terminal cleavage, thereby suggesting that this highly conserved residue regulates the balance between the two reactions.     

Proteomic analysis reveals differences between Vitis vinifera L. cv. Chardonnay and cv. Cabernet Sauvignon and their responses to water deficit and salinity

The impact of water deficit and salt stress on two important wine grape cultivars, Chardonnay and Cabernet Sauvignon, was investigated. Plants were exposed to increasing salinity and water deficit stress over a 16 d time period. Measurements of stem water potentials, and shoot and leaf lengths indicated that Chardonnay was more tolerant to these stresses than Cabernet Sauvignon. Shoot tips were harvested every 8 d for proteomic analysis using a trichloroacetic acid/acetone extraction protocol and two-dimensional gel electrophoresis. Proteins were stained with Coomassie Brilliant Blue, quantified, and then 191 unique proteins were identified using matrix-assisted laser desorption ionization time of flight/time of flight mass spectrometry. Peptide sequences were matched against both the NCBI nr and TIGR Vitis expressed sequence tag (EST) databases that had been implemented with all public Vitis sequences. Approximately 44% of the protein isoforms could be identified. Analysis of variance indicated that varietal difference was the main source of protein expression variation (40%). In stressed plants, reduction of the amount of proteins involved with photosynthesis, protein synthesis, and protein destination was correlated with the inhibition of shoot elongation. Many of the proteins up-regulated in Chardonnay were of unclassified or of unknown function, whereas proteins specifically up-regulated in Cabernet Sauvignon were involved in protein metabolism.     

A new method for the study of velopharyngeal function using gated magnetic resonance imaging

The purpose of this project was to assess the feasibility of imaging the velopharynx of adult volunteers during repetitive speech, using gated magnetic resonance imaging (MRI). Although a number of investigators have used conventional MRI in the study of the human vocal tract, the mismatch between the lengthy time necessary to acquire sufficiently detailed images and the rapidity of movement of the vocal tract during speech has forced investigators to acquire images either while the subject is at rest or during sustained utterances. The technique used here acquired a portion of each image during repetitive utterances, building the full image over multiple utterance cycles. The velopharyngeal portal was imaged on a 1.5-Tesla GE Signa LX 8.2 platform with gated fast spoiled gradient echo protocol. An external 1-Hertz trigger was fed to the cardiac gate. Subjects synchronized utterance of consonant-vowel syllables to a flashing light synchronized with the external trigger. Each acquisition of 30 phases per second at a single-slice location took 22 to 29 seconds. Four consonant-vowel syllables (/pa/, /ma/, /sa/, and /ka/) were evaluated. Subjects vocalized throughout the acquisition, beginning 5 to 6 seconds beforehand to establish a regular rhythm. Imaging of the velopharyngeal portal was performed for sagittal, velopharyngeal axial (aligned perpendicular to the "knee" of the velum), axial, and coronal planes. Volumes were obtained by sequential acquisition of six to 10 slices (each with 30 phases) in the axial or sagittal planes during repetition of the /pa/ syllable. Spatiotemporal volumes of the single-slice data were sectioned to provide time-motion images (analogous to M-mode echocardiograms). Three-dimensional dynamic volume renderings of palate motion were displayed interactively (Vortex; CieMed, Singapore). A method suitable for the collection and visualization of four-dimensional information regarding monosyllabic speech using gated MRI was developed. These techniques were applied to a population of adult volunteer subjects with no history of speech problems and two patients with a history of cleft lip and palate. The techniques allowed good real-time visualization of velopharyngeal anatomy during its entire range of motion and was also able to image pathology-specific anatomic differences in the subjects with cleft lip and cleft palate. These methods may be applicable to a wide spectrum of problems in speech physiology research and for clinical decision-making regarding surgery for speech and outcomes analysis.     

Lipid raft-dependent and -independent signaling through HLA-DR molecules

Lipid rafts are plasma membrane microdomains that are highly enriched in signaling molecules and that act as signal transduction platforms for many immune receptors. The involvement of these microdomains in HLA-DR-induced signaling is less well defined. We examined the constitutive presence of HLA-DR molecules in lipid rafts, their possible recruitment into these microdomains, and the role of these microdomains in HLA-DR-induced responses. We detected significant amounts of HLA-DR molecules in the lipid rafts of EBV(+) and EBV(-) B cell lines, monocytic cell lines, transfected HeLa cells, tonsillar B cells, and human monocytes. Localization of HLA-DR in these microdomains was unaffected by the deletion of the cytoplasmic domain of both the alpha and beta chains. Ligation of HLA-DR with a bivalent, but not a monovalent, ligand resulted in rapid tyrosine phosphorylation of many substrates, especially Lyn, and activation of ERK1/2 MAP kinase. However, the treatment failed to induce further recruitment of HLA-DR molecules into lipid rafts. The HLA-DR-induced signaling events were accompanied by the induction of cell-cell adhesion that could be inhibited by PTK and Lyn but not ERK1/2 inhibitors. Disruption of lipid rafts by methyl-beta-cyclodextrin (MbetaCD) resulted in the loss of membrane raft association with HLA-DR molecules, inhibition of HLA-DR-mediated protein tyrosine phosphorylation and cell-cell adhesion. MbetaCD did not affect the activation of ERK1/2, which was absent from lipid rafts. These results indicate that although all the HLA-DR-induced events studied are dependent on HLA-DR dimerization, some require the presence of HLA-DR molecules in lipid rafts, whereas others do not.     

Nucleus behavior during the closed mitosis of Tritrichomonas foetus

We present observations on the fine structure and the division process of the nucleus in the protist Tritrichomonas foetus, parasite of the urogenital tract of cattle. The nucleus was followed by immunofluorescence and electron microscopy during interphase and mitosis. Conventional karyotyping coupled to image processing and bright field Panotic staining were used to follow nucleus modifications, chromosome number and condensation pattern along the whole cell cycle. Confocal laser scanning microscopy (CLSM) using DNA fluorescent probes, followed by image processing in the SURF-Driver program, produced three-dimensional reconstruction data of the mitotic nucleus under each phase of the division process. Immunocytochemistry in thin-sections revealed the chromosome spatial arrangement after bromodeoxyuridine incorporation and immunogold labeling using anti-DNA monoclonal antibodies. Our results indicate that: (1) the nucleus assumes different size and shapes along mitosis: it appears oval in interphase, becoming lobed or concave in prophase, then undergoing torsion and constriction, displaying an 'S' shape (metaphase). Next, it becomes elongated and it is finally separated in two nuclei at the transition of anaphase to telophase; (2) T. foetus nucleus harbors five chromosomes; (3) chromosomes become condensed in a pre-mitotic phase; (4) the nucleolus persists during the mitosis.