On the Direction of Reading of Bacteriophage T4 Gene 43 (Deoxyribonucleic Acid Polymerase)

Amber (am) mutants of the two closely linked sites, B22 and C125, in bacteriophage T4 gene 43 [deoxyribonucleic acid (DNA) polymerase] synthesize in the nonpermissive (su(-)) Escherichia coli host gene 43 products which are devoid of DNA polymerase activity, but which retain a 3'-exonuclease activity. Diethylaminoethyl-cellulose chromatographic analysis of DNA polymerase and deoxyribonuclease activities from extracts of su(-) cells infected with single- and double-am mutants of T4 gene 43 showed that the exonuclease activity which is observed with amB22 is not seen with double mutants carrying, in addition to amB22, am mutations which map to the clockwise side of the B22 site on the circular genetic map of T4. Similarly, am mutations which map to the clockwise side of the C125 site abolish the exonuclease activity which is observed with an am mutant (amE4335) of this site. It was concluded that in these double mutants termination signals to the clockwise side of amB22 and amE4335 are encountered before the amB22 and amE4335 signals during translation of the messenger ribonucleic acid from T4 gene 43. Thus, it seems that the T4 DNA polymerase is synthesized in vivo in a direction which corresponds to a counterclockwise reading of gene 43.     

Grb7 SH2 domain structure and interactions with a cyclic peptide inhibitor of cancer cell migration and proliferation

Background

Human growth factor receptor bound protein 7 (Grb7) is an adapter protein that mediates the coupling of tyrosine kinases with their downstream signaling pathways. Grb7 is frequently overexpressed in invasive and metastatic human cancers and is implicated in cancer progression via its interaction with the ErbB2 receptor and focal adhesion kinase (FAK) that play critical roles in cell proliferation and migration. It is thus a prime target for the development of novel anti-cancer therapies. Recently, an inhibitory peptide (G7-18NATE) has been developed which binds specifically to the Grb7 SH2 domain and is able to attenuate cancer cell proliferation and migration in various cancer cell lines.

Results

As a first step towards understanding how Grb7 may be inhibited by G7-18NATE, we solved the crystal structure of the Grb7 SH2 domain to 2.1 Å resolution. We describe the details of the peptide binding site underlying target specificity, as well as the dimer interface of Grb 7 SH2. Dimer formation of Grb7 was determined to be in the μM range using analytical ultracentrifugation for both full-length Grb7 and the SH2 domain alone, suggesting the SH2 domain forms the basis of a physiological dimer. ITC measurements of the interaction of the G7-18NATE peptide with the Grb7 SH2 domain revealed that it binds with a binding affinity of K_d = ~35.7 μM and NMR spectroscopy titration experiments revealed that peptide binding causes perturbations to both the ligand binding surface of the Grb7 SH2 domain as well as to the dimer interface, suggesting that dimerisation of Grb7 is impacted on by peptide binding.

Conclusion

Together the data allow us to propose a model of the Grb7 SH2 domain/G7-18NATE interaction and to rationalize the basis for the observed binding specificity and affinity. We propose that the current study will assist with the development of second generation Grb7 SH2 domain inhibitors, potentially leading to novel inhibitors of cancer cell migration and invasion.     

The small regulatory RNA molecule MicA is involved in <Emphasis Type="Italic">Salmonella enterica</Emphasis> serovar Typhimurium biofilm formation

Background  

LuxS is the synthase enzyme of the quorum sensing signal AI-2. In Salmonella Typhimurium, it was previously shown that a luxS deletion mutant is impaired in biofilm formation. However, this phenotype could not be complemented by extracellular addition of quorum sensing signal molecules.     

Functional characterisation of the regulation of CAAT enhancer binding protein alpha by GSK-3 phosphorylation of Threonines 222/226

Background  

Glycogen Synthase Kinase-3 (GSK3) activity is repressed following insulin treatment of cells. Pharmacological inhibition of GSK3 mimics the effect of insulin on Phosphoenolpyruvate Carboxykinase (PEPCK), Glucose-6 Phosphatase (G6Pase) and IGF binding protein-1 (IGFBP1) gene expression. CAAT/enhancer binding protein alpha (C/EBPα) regulates these gene promoters in liver and is phosphorylated on two residues (T222/T226) by GSK3, although the functional outcome of the phosphorylation has not been established. We aimed to establish whether CEBPα is a link between GSK3 and these gene promoters.     

Multiple duplications of yeast hexose transport genes in response to selection in a glucose-limited environment

When microbes evolve in a continuous, nutrient-limited environment, natural selection can be predicted to favor genetic changes that give cells greater access to limiting substrate. We analyzed a population of baker's yeast that underwent 450 generations of glucose-limited growth. Relative to the strain used as the inoculum, the predominant cell type at the end of this experiment sustains growth at significantly lower steady-state glucose concentrations and demonstrates markedly enhanced cell yield per mole glucose, significantly enhanced high-affinity glucose transport, and greater relative fitness in pairwise competition. These changes are correlated with increased levels of mRNA hybridizing to probe generated from the hexose transport locus HXT6. Further analysis of the evolved strain reveals the existence of multiple tandem duplications involving two highly similar, high- affinity hexose transport loci, HXT6 and HXT7. Selection appears to have favored changes that result in the formation of more than three chimeric genes derived from the upstream promoter of the HXT7 gene and the coding sequence of HXT6. We propose a genetic mechanism to account for these changes and speculate as to their adaptive significance in the context of gene duplication as a common response of microorganisms to nutrient limitation.      

Acceptor specificity of the human leukocyte alpha3 fucosyltransferase: role of FucT-VII in the generation of selectin ligands

The alpha3 fucosyltransferase, FucT-VII, is one of the key glycosyltransferases involved in the biosynthesis of the sialyl Lewis X (sLex) antigen on human leukocytes. The sialyl Lewis X antigen (NeuAcalpha(2-3)Galbeta(1-4)[Fucalpha(1-3)]GlcNAc-R) is an essential component of the recruitment of leukocytes to sites of inflammation, mediating the primary interaction between circulating leukocytes and activated endothelium. In order to characterize the enzymatic properties of the leukocyte alpha3 fucosyltransferase FucT-VII, the enzyme has been expressed in Trichoplusia ni insect cells. The enzyme is capable of synthesizing both sLexand sialyl-dimeric-Lexstructures in vitro , from 3'-sialyl-lacNAc and VIM-2 structures, respectively, with only low levels of fucose transfer observed to neutral or 3'-sulfated acceptors. Studies using fucosylated NeuAcalpha(2-3)-(Galbeta(1- 4)GlcNAc)3-Me acceptors demonstrate that FucT-VII is able to synthesize both di-fucosylated and tri-fucosylated structures from mono- fucosylated precursors, but preferentially fucosylates the distal GlcNAc within a polylactosamine chain. Furthermore, the rate of fucosylation of the internal GlcNAc residues is reduced once fucose has been added to the distal GlcNAc. These results indicate that FucT-VII is capable of generating complex selectin ligands, in vitro , however the order of fucose addition to the lactosamine chain affects the rate of selectin ligand synthesis.      

ERRATUM: New Body Mass Estimates for Omomys carteri,a Middle Eocene Primate From North America. Am J Phys Anthropol 109:41–52.

Payseur BA, Covert HA, Vinyard CJ, Dagosto M. 1999. New Body Mass Estimates for Omomys carteri, a Middle Eocene Primate From North America. Am J Phys Anthropol 109:41–52. This article included an incomplete Table 2. The final two columns, showing “Intercept” and “SEE” data were omitted. The complete Table 2, with these two columns included, is provided below.     

High definition bronchoscopy: a randomized exploratory study of diagnostic value compared to standard white light bronchoscopy and autofluorescence bronchoscopy

Background

Videobronchoscopy is an essential diagnostic procedure for evaluation of the central airways and pivotal for the diagnosis and staging of lung cancer. Technological improvements have resulted in high definition (HD) images with advanced real time image enhancement techniques (i-scan).

Objectives

In this study we aimed to explore the sensitivity of HD+ i-scan bronchoscopy for detection of epithelial changes like vascular abnormalities and suspicious preinvasive lesions, and tumors.

Methods

In patients scheduled for a therapeutic or diagnostic procedure under general anesthesia videos of the bronchial tree were made using 5 videobronchoscopy modes in random order: normal white light videobronchoscopy (WLB), HD-bronchoscopy (HD), HD bronchoscopy with surface enhancement technique (i-scan1), HD with surface- and tone enhancement technique (i-scan2) and dual mode autofluorescence videobronchoscopy (AFB). The videos were scored in random order by two independent and blinded expert bronchoscopists.

Results

In 29 patients all videos were available for analysis. Vascular abnormalities were scored most frequently in HD + i-scan2 bronchoscopy (1.33 ± 0.29 abnormal or suspicious sites per patient) as compared to 0.12 ± 0.05 site for AFB (P = 0.003). Sites suspicious for preinvasive lesions were most frequently reported using AFB (0.74 ± 0.12 sites per patient) as compared to 0.17 ± 0.06 for both WLB and HD bronchoscopy (P = 0.003). Tumors were detected equally by all modalities. The preferred modality was HD bronchoscopy with i-scan (tone- plus surface and surface enhancement in respectively 38% and 35% of cases P = 0.006).

Conclusions

This study shows that high definition bronchoscopy with image enhancement technique may result in better detection of subtle vascular abnormalities in the airways. Since these abnormalities may be related to preneoplastic lesions and tumors this is of clinical relevance. Further investigations using this technique relating imaging to histology are warranted.     

The BioLexicon: a large-scale terminological resource for biomedical text mining

Background

Due to the rapidly expanding body of biomedical literature, biologists require increasingly sophisticated and efficient systems to help them to search for relevant information. Such systems should account for the multiple written variants used to represent biomedical concepts, and allow the user to search for specific pieces of knowledge (or events) involving these concepts, e.g., protein-protein interactions. Such functionality requires access to detailed information about words used in the biomedical literature. Existing databases and ontologies often have a specific focus and are oriented towards human use. Consequently, biological knowledge is dispersed amongst many resources, which often do not attempt to account for the large and frequently changing set of variants that appear in the literature. Additionally, such resources typically do not provide information about how terms relate to each other in texts to describe events.

Results

This article provides an overview of the design, construction and evaluation of a large-scale lexical and conceptual resource for the biomedical domain, the BioLexicon. The resource can be exploited by text mining tools at several levels, e.g., part-of-speech tagging, recognition of biomedical entities, and the extraction of events in which they are involved. As such, the BioLexicon must account for real usage of words in biomedical texts. In particular, the BioLexicon gathers together different types of terms from several existing data resources into a single, unified repository, and augments them with new term variants automatically extracted from biomedical literature. Extraction of events is facilitated through the inclusion of biologically pertinent verbs (around which events are typically organized) together with information about typical patterns of grammatical and semantic behaviour, which are acquired from domain-specific texts. In order to foster interoperability, the BioLexicon is modelled using the Lexical Markup Framework, an ISO standard.

Conclusions

The BioLexicon contains over 2.2 M lexical entries and over 1.8 M terminological variants, as well as over 3.3 M semantic relations, including over 2 M synonymy relations. Its exploitation can benefit both application developers and users. We demonstrate some such benefits by describing integration of the resource into a number of different tools, and evaluating improvements in performance that this can bring.