Plant regeneration through culture of isolated microspores of Triticum aestivum L.

Wheat microspores mechanically isolated from the anthers before culture and isolated from the anthers during the hole culture period in a chemically defined medium resulted in proembryos, embryos and finally plants. Of the four genotypes included, all responded with proembryos, and the two spring wheats Ciano and Walter gave rise to macroscopic embryos and plants. The frequency of embryo regeneration and the frequency of albino plants in both Ciano and Walter was in accordance with previously obtained results with anther culture derived material.Abbreviations 2,4-d 2,4-dichlorophenoxy acetic acid - NAA 1-naphthaleneacetic acid     

Two frameshift mutations in the cystic fibrosis gene

Cystic fibrosis (CF) is a recessive disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. We have identified in exon 7 two frameshift mutations, one caused by a two-nucleotide insertion and the other caused by a one-nucleotide deletion; these mutations--CF1154insTC and CF1213delT, respectively, are predicted to shift the reading frame of the protein and to introduce UAA(ochre) termination codons at residues 369 and 368.     

Mobility of normal and virus-transformed cells in cellular aggregates

The mobility of embryonic chick cells and cells of four established cell lines was examined in cellular aggregates. This was done by preparing aggregates of unlabeled cells and allowing cells of the same type, but prelabeled with [3H]thymidine, to adhere to the surface of the aggregates. After 2-1/2 days in agitated liquid culture the positions of the labeled cells within the aggregates were determined by autoradiographic techniques. Since the labeled and unlabeled cells were otherwise identical, the degree of penetration of the labeled cells into the aggregates was taken as a measure of the mixing or mobility of cells in the aggregate. With this procedure, embryonic chick liver, heart, and neural retina cells were found to move an average of 2.12, 2.68, and 4.00 cell diameters inward, respectively. Mouse fibroblast BALB/c 3T3 cells moved an average of 1.13 cell diameters inward, while Simian virus 40 (SV40)-transformed BALB/c 3T3 cells moved as much as 8.80 cell diameters inward, indicating that cells of the malignant SV40-transformed line were considerably more mobile than the corresponding nonmalignant 3T3 cells. In contrast, cells of the hamster fibroblast line NIL B moved 4.17 cell diameters in 2-1/2 days, while SV40-transformed NIL B cells moved 3.00 cell diameters in the same time. It was therefore concluded that infection with oncogenic viruses does not necessarily result in increased cellular mobility.     

Precision mapping of the human O‐GalNAc glycoproteome through SimpleCell technology

Glycosylation is the most abundant and diverse posttranslational modification of proteins. While several types of glycosylation can be predicted by the protein sequence context, and substantial knowledge of these glycoproteomes is available, our knowledge of the GalNAc‐type O‐glycosylation is highly limited. This type of glycosylation is unique in being regulated by 20 polypeptide GalNAc‐transferases attaching the initiating GalNAc monosaccharides to Ser and Thr (and likely some Tyr) residues. We have developed a genetic engineering approach using human cell lines to simplify O‐glycosylation (SimpleCells) that enables proteome‐wide discovery of O‐glycan sites using ‘bottom‐up’ ETD‐based mass spectrometric analysis. We implemented this on 12 human cell lines from different organs, and present a first map of the human O‐glycoproteome with almost 3000 glycosites in over 600 O‐glycoproteins as well as an improved NetOGlyc4.0 model for prediction of O‐glycosylation. The finding of unique subsets of O‐glycoproteins in each cell line provides evidence that the O‐glycoproteome is differentially regulated and dynamic. The greatly expanded view of the O‐glycoproteome should facilitate the exploration of how site‐specific O‐glycosylation regulates protein function.     

Mechanisms Preserving Insulin Action during High Dietary Fat Intake

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Indirect intracoronary delivery of adenovirus encoding adenylyl cyclase increases left ventricular contractile function in mice

We performed indirect intracoronary delivery of adenovirus vectors in mice and explored techniques including hypothermia and pharmacological means to increase cardiac gene transfer. Mice were maintained in a normothermic state or cooled to 25 degrees C. The aorta or both the pulmonary artery and aorta were clamped while a needle was advanced into the left ventricular cavity to deliver adenovirus vectors encoding enhanced green fluorescent protein (EGFP) or murine adenylyl cyclase type VI (AC(VI)) with saline, sodium nitroprusside, acetylcholine, or serotonin. Clamping was maintained for 30 s (normothermia) or 2 min (25 degrees C) after adenovirus administration. Mice were killed 7 or 21 days later, and hearts were examined for EGFP expression. Compared with clamping the aorta alone and with no cooling, gene transfer was increased as follows: 1) 1.3-fold with hypothermia to extend dwell time; 2) 4.5-fold by clamping the aorta and the pulmonary artery; 3) 11.4-fold with nitroprusside administration; 4) 11.8-fold with serotonin addition, and 5) 14.3-fold with acetylcholine delivery. Gene expression remained substantial at 21 days, and no significant inflammatory response was seen. Efficacy of the method was tested by performing gene transfer of adenovirus encoding AC(VI). Fourteen days after gene transfer, hearts isolated from mice that received adenovirus encoding AC(VI) showed increased contractile function. Indirect intracoronary delivery of adenovirus vectors in mice is associated with efficient cardiac gene transfer and increased left ventricular function after AC(VI) gene transfer.     

Putative in vitro expressed gene fragments unique to Mycobacterium avium subspecies paratuberculosis

By a suppression subtractive hybridization based method, nine novel Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) fragments of between 318 and 596 bp have been identified and characterized. Database search revealed little or no similarity with other mycobacteria. The uniqueness and diagnostic potential of seven of these fragments in relation to M. paratuberculosis closest relative Mycobacterium avium subsp. avium (M. avium) was confirmed by species-specific PCR and Southern blot. Furthermore, RT-PCR indicated that eight of the nine fragments originate from areas of the genome that are expressed in vitro.     

The motion of a single molecule,the lambda-receptor,in the bacterial outer membrane

Using optical tweezers and single particle tracking, we have revealed the motion of a single protein, the lambda-receptor, in the outer membrane of living Escherichia coli bacteria. We genetically modified the lambda-receptor placing a biotin on an extracellular site of the receptor in vivo. The efficiency of this in vivo biotinylation is very low, thus enabling the attachment of a streptavidin-coated bead binding specifically to a single biotinylated lambda-receptor. The bead was used as a handle for the optical tweezers and as a marker for the single particle tracking routine. We propose a model that allows extraction of the motion of the protein from measurements of the mobility of the bead-molecule complex; these results are equally applicable to analyze bead-protein complexes in other membrane systems. Within a domain of radius approximately 25 nm, the receptor diffuses with a diffusion constant of (1.5 +/- 1.0) x 10(-9) cm(2)/s and sits in a harmonic potential as if it were tethered by an elastic spring of spring constant of ~1.0 x 10(-2) pN/nm to the bacterial membrane. The purpose of the protein motion might be to facilitate transport of maltodextrins through the outer bacterial membrane.