Identical Mr 70,000 S6 kinase is activated biphasically by epidermal growth factor: a phosphopeptide that characterizes the late phase.

Mitogenic stimulation of quiescent mouse 3T3 cells with epidermal growth factor leads to biphasic S6 kinase activation. The kinases present in both phases of the response have been purified from 32P-labeled cells and shown to contain a phosphoprotein of equivalent Mr 70,000. Chromatographic analysis of the purified S6 kinases on a Mono Q column reveals that (i) all 32P-labeled protein coelutes with S6 kinase activity, (ii) only those fractions containing S6 kinase autophosphorylate, (iii) autophosphorylation is restricted to a single Mr 70,000 protein, and (iv) the extent of autophosphorylation directly parallels the degree of S6 kinase activation. Analysis of the two autophosphorylated S6 kinases by two-dimensional tryptic phosphopeptide mapping indicates that they are the same protein. Both in vivo 32P-labeled S6 kinases contain phosphoserine and phosphothreonine but no detectable phosphotyrosine. Two-dimensional tryptic peptide maps of the in vivo 32P-labeled S6 kinases are essentially identical, except for a single qualitative change in the late-phase S6 kinase.     

In vivo and ex vivo sentinel node mapping does not identify the same lymph nodes in colon cancer

Introduction

Identification of lymph nodes and pathological analysis is crucial for the correct staging of colon cancer. Lymph nodes that drain directly from the tumor area are called “sentinel nodes” and are believed to be the first place for metastasis. The purpose of this study was to perform sentinel node mapping in vivo with indocyanine green and ex vivo with methylene blue in order to evaluate if the sentinel lymph nodes can be identified by both techniques.

Methods

Patients with colon cancer UICC stage I–III were included from two institutions in Denmark from February 2015 to January 2016. In vivo sentinel node mapping with indocyanine green during laparoscopy and ex vivo sentinel node mapping with methylene blue were performed in all patients.

Results

Twenty-nine patients were included. The in vivo sentinel node mapping was successful in 19 cases, and ex vivo sentinel node mapping was successful in 13 cases. In seven cases, no sentinel nodes were identified. A total of 51 sentinel nodes were identified, only one of these where identified by both techniques (2.0%). In vivo sentinel node mapping identified 32 sentinel nodes, while 20 sentinel nodes were identified by ex vivo sentinel node mapping. Lymph node metastases were found in 10 patients, and only two had metastases in a sentinel node.

Conclusion

Placing a deposit in relation to the tumor by indocyanine green in vivo or of methylene blue ex vivo could only identify sentinel lymph nodes in a small group of patients.

     

Beta -Globin mRNA decay in erythroid cells: UG site-preferred endonucleolytic cleavage that is augmented by a premature termination codon

Previous work showed that human beta-globin mRNAs harboring a premature termination codon are degraded in the erythroid tissues of mice to products that lack sequences from the mRNA 5' end but contain a 5' cap-like structure. Whether these decay products are the consequence of endonucleolytic or 5'-to-3' exonucleolytic activity is unclear. We report that this beta-globin mRNA decay pathway is recapitulated in cultured mouse erythroleukemia (MEL) cells and targets nonsense-free mRNA to a lesser extent than nonsense-containing mRNA. S1 nuclease mapping and primer extension demonstrated that 70-80% of decay product 5' ends contain a UG dinucleotide. Detection of upstream counterparts of these decay products indicates that they are generated by endonucleolytic activity. Both crude and partially purified polysome extracts prepared from MEL cells contain an endonucleolytic activity that generates decay products comparable to those observed in vivo. These data suggest that an endonuclease with preference for UG dinucleotides is involved in the degradation of nonsense-containing and, to a lesser extent, nonsense-free human beta-globin mRNAs in mouse erythroid cells.     

In vivo identification of human cortical areas using high-resolution MRI: an approach to cerebral structure-function correlation

Understanding the relationship between the structural and functional organization of the human brain is one of the most important goals of neuroscience. Individual variability in brain structure means that it is essential to obtain this information from the same subject. To date, this has been almost impossible. Even though noninvasive functional imaging techniques such as functional MRI (fMRI) are now commonplace, there is no complementary noninvasive structural technique. We present an in vivo method of examining the detailed neuroanatomy of any individual, which can then be correlated with that individual's own functional results. This method utilizes high-resolution structural MRI to identify distinct cortical regions based on cortical lamination structure. We demonstrate that the observed MR lamination patterns relate to myeloarchitecture through a correlation of histology with MRI. In vivo high-resolution MRI studies identify striate cortex, as well as visual area V5, in four individuals, as defined by using fMRI. The anatomical identification of a cortical area (V5MT) outside of striate cortex is a significant advance, proving it possible to identify extra-striate cortical areas and demonstrating that in vivo structural mapping of the human cerebral cortex is possible.     

Construction of the physical map for three loci in chromosome band 13q14: comparison to the genetic map.

Pulsed-field gel electrophoresis (PFGE) and deletion mapping are being used to construct a physical map of the long arm of human chromosome 13. The present study reports a 2700-kilobase (kb) Not I long-range restriction map encompassing the 13q14-specific loci D13S10, D13S21, and D13S22, which are detected by the cloned DNA markers p7D2, pG24E2.4, and pG14E1.9, respectively. Analysis of a panel of seven cell lines that showed differential methylation at a Not I site between D13S10 and D13S21 proved physical linkage of the two loci to the same 875-kb Not I fragment. D13S22 mapped to a different Not I fragment, precluding the possibility that D13S22 is located between D13S10 and D13S21. PFGE analysis of Not I partial digests placed the 1850-kb Not I fragment containing D13S22 immediately adjacent to the 875-kb fragment containing the other two loci. The proximal rearrangement breakpoint in a cell line carrying a del13(q14.1q21.2) was detected by D13S21 but not by D13S10, demonstrating that D13S21 lies proximal to D13S10. Quantitative analysis of hybridization signals of the three DNA probes to DNA from the same cell line indicated that only D13S10 was deleted, establishing the order of these loci to be cen-D13S22-D13S21-D13S10-tel. Surprisingly, this order was estimated to be 35,000 times less likely than that favored by genetic linkage analysis.     

Mapping nucleosome position at single base-pair resolution by using site-directed hydroxyl radicals.

A base-pair resolution method for determining nucleosome position in vitro has been developed to com- plement existing, less accurate methods. Cysteaminyl EDTA was tethered to a recombinant histone octamer via a mutant histone H4 with serine 47 replaced by cysteine. When assembled into nucleosome core particles, the DNA could be cut site specifically by hydroxyl radical-catalyzed chain scission by using the Fenton reaction. Strand cleavage occurs mainly at a single nucleotide close to the dyad axis of the core particle, and assignment of this location via the symmetry of the nucleosome allows base-pair resolution mapping of the histone octamer position on the DNA. The positions of the histone octamer and H3H4 tetramer were mapped on a 146-bp Lytechinus variegatus 5S rRNA sequence and a twofold-symmetric derivative. The weakness of translational determinants of nucleosome positioning relative to the overall affinity of the histone proteins for this DNA is clearly demonstrated. The predominant location of both histone octamer and H3H4 tetramer assembled on the 5S rDNA is off center. Shifting the nucleosome core particle position along DNA within a conserved rotational phase could be induced under physiologically relevant conditions. Since nucleosome shifting has important consequences for chromatin structure and gene regulation, an approach to the thermodynamic characterization of this movement is proposed. This mapping method is potentially adaptable for determining nucleosome position in chromatin in vivo.     

A protein kinase from Xenopus eggs specific for ribosomal protein S6.

A protein kinase specific for ribosomal protein S6 has been purified from eggs of Xenopus laevis. As visualized on a silver-stained polyacrylamide gel, the major protein in the preparation migrated with a Mr of 90,000. Incubation of the enzyme preparation with [gamma-32P]ATP led to phosphorylation of this protein on serine residues. Upon glycerol gradient centrifugation, the S6 kinase activity and the Mr 90,000 protein both sedimented with a Mr of 50,000-55,000. Two-dimensional gel electrophoresis demonstrated that up to 4-5 phosphate groups per S6 molecule could be incorporated with this enzyme in vitro, and two-dimensional peptide mapping demonstrated that the phosphopeptides from S6 labeled in vitro with the enzyme comigrated with those from highly phosphorylated S6 labeled in vivo in response to progesterone treatment. The purified S6 protein kinase did not phosphorylate at a significant rate ribosomal protein S10, histone H1, histone H4, mixed histones, casein, or phosvitin, indicating a high degree of substrate specificity. These results indicate that activation of a single S6 protein kinase may be sufficient to account for increased S6 phosphorylation after a growth stimulus.     

Prolonged signal-averaged P-wave duration as an intermediate phenotype for familial atrial fibrillation.

OBJECTIVES: This study sought to perform a genome-wide linkage analysis in a large atrial fibrillation (AF) kindred using AF and abnormally prolonged signal-averaged (SA) P-wave duration as the phenotype. BACKGROUND: Although inherited forms of AF exist, phenotypic complexity has limited efforts to ascertain mutation carriers and thus identify causal genes. The identification of intermediate or endophenotypes may accelerate this effort. METHODS: A genome-wide linkage analysis was performed in a 4-generation AF kindred of 27 individuals, 8 with AF documented by electrocardiogram. The analysis was performed using AF as the phenotype, and repeated using an abnormally prolonged SA P-wave duration as the phenotype. RESULTS: Linkage analysis and fine mapping generated a maximum multipoint logarithm of the odds (LOD) score of 3.0 at chromosome 5p15 between markers D5S406 and D5S635. Importantly, 8 heterozygous carriers had a prolonged SA P-wave (203 +/- 21 ms) compared with 17 noncarriers (116 +/- 12 ms, p < 0.00001). Using prolonged SA P-wave (conventionally defined as >155 ms) as an endophenotype, a maximum LOD score of 3.6 was obtained in the same region of chromosome 5p15, a span of 5.75 centi-Morgans. CONCLUSIONS: In a large AF kindred, we have identified a novel AF locus on chromosome 5p15 and shown that affected individuals with AF and mutation carriers can be identified by a prolonged SA P-wave duration. Importantly, identification of an endophenotype in this kindred not only aided ascertainment of additional family members but also increased the LOD score, providing increased support for linkage at this locus. Identification of the causal gene, mapped to chromosome 5p15, will advance our understanding of the molecular basis of AF.     

Site-specific mutations alter in vitro factor binding and change promoter expression pattern in transgenic plants.

The 35S promoter of cauliflower mosaic virus (CaMV) is able to confer high-level gene expression in most organs of transgenic plants. A cellular factor from pea and tobacco leaf tissue, which recognizes nucleotides in a tandemly repeated TGACG motif at the -75 region of this promoter, has been detected by DNase I footprinting and gel retardation assays. This factor is named activation sequence factor 1 (ASF-1). A cellular factor binding to the two TGACG motifs can also be detected in tobacco root extracts. Mutations at these motifs inhibit binding of ASF-1 to the 35S promoter in vitro. When examined in transgenic tobacco, these mutations cause a 50% drop in leaf expression of the 35S promoter. In addition, these same mutations attenuate stem and root expression of the 35S promoter about 5- to 10-fold when compared to the level of expression in leaf. In contrast, mutations at two adjacent CCAAT-box-like sequences have no dramatic effect on promoter activity in vivo. A 21-base-pair element containing the two TGACG motifs is sufficient for binding of ASF-1 in vitro when inserted in a green-tissue-specific promoter. In vivo, the insertion of an ASF-1 binding site caused high levels of expression in root. Thus, a single factor binding site that is defined by site-specific mutations is shown to be sufficient to alter the expression pattern of promoters in vivo.     

Mapping knowledge domains: characterizing PNAS

A review of data mining and analysis techniques that can be used for the mapping of knowledge domains is given. Literature mapping techniques can be based on authors, documents, journals, words, and/or indicators. Most mapping questions are related to research assessment or to the structure and dynamics of disciplines or networks. Several mapping techniques are demonstrated on a data set comprising 20 years of papers published in PNAS. Data from a variety of sources are merged to provide unique indicators of the domain bounded by PNAS. By using funding source information and citation counts, it is shown that, on an aggregate basis, papers funded jointly by the U.S. Public Health Service (which includes the National Institutes of Health) and non-U.S. government sources outperform papers funded by other sources, including by the U.S. Public Health Service alone. Grant data from the National Institute on Aging show that, on average, papers from large grants are cited more than those from small grants, with performance increasing with grant amount. A map of the highest performing papers over the 20-year period was generated by using citation analysis. Changes and trends in the subjects of highest impact within the PNAS domain are described. Interactions between topics over the most recent 5-year period are also detailed.     

Changes in the propagation pattern within the conduction channel during sinus rhythm and ventricular tachycardia demonstrated by non-contact mapping: role of late potential activity

Sustained monomorphic ventricular tachycardia (VT) in patients with a previous myocardial infarction is due to re-entry mechanism in areas of slow conduction. The recognition of the pathogenic mechanism and the characterization of the activation pathway are usually obtained by indirect measures with entrainment mapping and pacing manoeuvres. We studied a 61-years-old patient with a history of previous inferior myocardial infarction and we provided the in vivo direct visualization of the critical components of re-entry circuit by non-contact mapping. VT circuit entrance, central pathway, and exit were characterized during the same beat by virtual electrodes and visualized on a three-dimensional map both during sinus rhythm, ongoing VT, and pacemapping. The analysis demonstrated an activation of the conductive channel in opposite directions during the sinus rhythm and ventricular tachycardia. Late potentials during sinus rhythm turned into mid-diastolic activity during VT; non-contact mapping allowed the ablation procedure to be performed in sinus rhythm, targeting the central pathway of the conducting channel and the abolition of VT inducibility.     

Synthesis of Simian Virus 40 DNA in Isolated Nuclei

Nuclei isolated from African green monkey kidney cells infected with simian virus 40 at different times after infection maintain in vitro the same temporal sequence of host and viral DNA synthesis as that seen in intact cells. The viral DNA synthesized by the nuclei of cells previously infected for 32-35 hr was characterized by centrifugation through neutral and alkaline sucrose gradients, and by isopycnic banding in a propidium iodide-cesium chloride gradient. DNA synthesis in this system is maintained for only 4-5 min. Neutral sucrose gradient analysis showed that most of the radioactivity is associated with the replicative intermediate of simian virus 40 DNA and the rest sediments at 5-7 S. Alkaline gradient analysis showed that 50-60% of the radioactivity sediments as 3-7S fragments, and the rest between 7 and 16 S. Pulsechase experiments showed that in this system 3-7S fragments do not mature into long chains. A model is presented to explain the failure of these fragments to join into long chains in this in vitro nuclear system.