Expression in cowpea seedlings of chimeric transgenes after electroporation into seed-derived embryos

Summary Cowpea (Vigna unguiculata Walp) embryos mechanically isolated from mature seeds and incubated in the presence of plasmid DNA harboring chimeric gus genes were shown to germinate into seedlings expressing -glucuronidase activity in a variety of tissues, including the apical meristem. Embryo electroporation in the presence of DNA and protectants such as spermine and Lipofectin^TM increased both the proportion of embryo-derived seedlings expressing the chimeric gene and the level of gene expression. Microscopic observations of thin sections showed that the blue crystals representing the end product of transgene activity on X-glu were exclusively located inside the treated cells. Histological localization of the blue dye crystals varied with the promoter used to drive the transgene.     

Monoclonal antibodies to Escherichia coli ribosomal proteins L9 and L10. Effects on ribosome function and localization of L9 on the surface of the 50 S ribosomal subunit

Monoclonal antibodies against Escherichia coli ribosomal proteins L9 and L10 were obtained and their specificity confirmed by Western blot analysis of total ribosomal protein. This was particularly important for the L9 antibody, since the immunizing antigen mixture contained predominantly L11. Each antibody recognized both 70 S ribosomes and 50 S subunits. Affinity-purified antibodies were tested for their effect on in vitro assays of ribosome function. Anti-L10 and anti-L9 inhibited poly(U)-directed polyphenylalanine synthesis almost completely. The antibodies had no effect on subunit association or dissociation and neither antibody inhibited peptidyltransferase activity. Both antibodies inhibited the binding of the ternary complex that consisted of aminoacyl-tRNA, guanylyl beta, gamma-methylenediphosphonate, and elongation factor Tu, and the binding of elongation factor G to the ribosome. The intact antibodies were more potent inhibitors than the Fab fragments. In contrast to the previously established location of L10 at the base of the L7/L12 stalk near the factor-binding site, the site of anti-L9 binding to 50 S subunits was shown by immune electron microscopy to be on the L1 lateral protuberance opposite the L7/L12 stalk as viewed in the quasisymmetric projection. The inhibition of factor binding by both antibodies, although consistent with established properties of L10 in the ribosome, suggests a long range effect on subunit structure that is triggered by the binding of anti-L9.     

Structure of ebenone,a possible biogenetic precursor of elliptinone,from Diospyros ebenum

The isolation and determination of the structure of ebenone, a new biogenetically significant β-naphthyl-1,4-naphthaquinone derivative, from Diospyros ebenum is reported.     

Chitosan in Surface Modification for Bone Tissue Engineering Applications

Traditional methods of bone defect repair include autografts, allografts, surgical reconstruction, and metal implants that have several disadvantages such as donor site morbidity, rejection, risk of disease transmission, and repetitive surgery. Biomaterial‐based bone reconstructions can, therefore, be an efficient alternative due to the inherent properties of the materials. Chitosan (CS), the deacetylated form of chitin, is a biopolymer having a wide array of applicability in regenerative tissue applications owing to its biocompatible, in vitro degradative and bioresorbable nature. Extensive studies are being carried out using CS to augment the properties of the already existing methods and to also improve the applicability of CS‐based biocomposites in bone tissue repair. In this review, the suitability of CS as a surface modifier has been discussed in detail for the already existing implants, surface modifications of CS‐based natural biocomposites for bone tissue regeneration, and the wide range of techniques that can introduce these modifications. CS, being a natural polymer, possesses advantageous properties including surface modifier that makes it a suitable candidate for bone regeneration, and further research to investigate its osteogenic potential in vivo along with the molecular and signaling mechanisms involved in bone regeneration can aid in expanding its applicability in clinical trials.     

SeMoP: a new computational strategy for the unrestricted search for modified peptides using LC-MS/MS data

A novel computational approach, termed Search for Modified Peptides (SeMoP), for the unrestricted discovery and verification of peptide modifications in shotgun proteomic experiments using low resolution ion trap MS/MS spectra is presented. Various peptide modifications, including post-translational modifications, sequence polymorphisms, as well as sample handling-induced changes, can be identified using this approach. SeMoP utilizes a three-step strategy: (1) a standard database search to identify proteins in a sample; (2) an unrestricted search for modifications using a newly developed algorithm; and (3) a second standard database search targeted to specific modifications found using the unrestricted search. This targeted approach provides verification of discovered modifications and, due to increased sensitivity, a general increase in the number of peptides with the specific modification. The feasibility of the overall strategy has been first demonstrated in the analysis of 65 plasma proteins. Various sample handling induced modifications, such as beta-elimination of disulfide bridges and pyrocarbamidomethylation, as well as biologically induced modifications, such as phosphorylation and methylation, have been detected. A subsequent targeted Sequest search has been used to verify selected modifications, and a 4-fold increase in the number of modified peptides was obtained. In a second application, 1367 proteins of a cervical cancer cell line were processed, leading to detection of several novel amino acid substitutions. By conducting the search against a database of peptides derived from proteins with decoy sequences, a false discovery rate of less than 5% for the unrestricted search resulted. SeMoP is shown to be an effective and easily implemented approach for the discovery and verification of peptide modifications.     

Insufficient rest or sleep and its relation to cardiovascular disease, diabetes and obesity in a national, multiethnic sample

Background

A new question on insufficient rest/sleep was included in the 2008 Behavioral Risk Factor Surveillance System (BRFSS) for the 50 states, District of Columbia, and three US territories. No previous study, however, has examined perceived insufficient rest/sleep in relation to cardiovascular disease (CVD) or diabetes mellitus. We examined the association between self-reported insufficient rest/sleep and CVD, diabetes, and obesity in a contemporary sample of US adults.

Methods

Multiethnic, nationally representative, cross-sectional survey (2008 BRFSS) participants were >20 years of age (n = 372, 144, 50% women). Self-reported insufficient rest/sleep in the previous month was categorized into four groups: zero, 1–13, 14–29, and 30 days. There were five outcomes: 1) any CVD, 2) coronary heart disease (CHD), 3) stroke, 4) diabetes mellitus, and 5) obesity (body mass index≥30 kg/m²). We employed multivariable logistic regression to calculate odds ratio (OR), (95% confidence interval (CI), of increasing categories of insufficient rest/sleep, taking zero days of insufficient rest/sleep as the referent category.

Principal Findings

Insufficient rest/sleep was found to be associated with 1) any CVD, 2) CHD, 3) stroke, 4) diabetes mellitus, and 5) obesity, in separate analyses. Compared to those reporting zero days of insufficient sleep (referent), the OR (95% CI) associated with all 30 days of insufficient sleep was 1.67 (1.55–1.79) for any cardiovascular disease, 1.69(1.56–1.83) for CHD, 1.51(1.36–1.68) for stroke, 1.31(1.21–1.41) for diabetes, and 1.51 (1.43–1.59) for obesity.

Conclusions

In a multiethnic sample of US adults, perceived insufficient rest/sleep was found to be independently associated with CHD, stroke, diabetes mellitus and obesity.