SV40 immortalizes myogenic cells: DNA synthesis and mitosis in differentiating myotubes

Primary skeletal muscle myoblasts have a limited proliferative capacity in cell culture and cease to proliferate after several passages. We examined the effects of several oncogenes on the immortalization and differentiation of primary cultures of rat skeletal muscle myoblasts. Retroviruses containing a SV40 large T antigen (LT) gene very efficiently immortalize myogenic cells. The immortalized cell lines retain a very high differentiation capacity and form, in the appropriate culture conditions, a very dense network of muscle fibers. As in primary culture, cell fusion is associated with the synthesis of large amounts of muscle-specific proteins. However, unlike normal myoblasts (and previously established myogenic cell lines), nuclei in the multinucleated fibers of SV40-immortalized cells synthesize DNA and enter mitosis. Thus, withdrawal from DNA synthesis is not obligatory for cell fusion and biochemical differentiation. Using a retrovirus coding for a temperature-sensitive SV40 LT, myogenic cell lines were produced in which the SV40 LT could be inactivated by a shift from 33 degrees C to 39 degrees C. The inactivation of LT induced massive cell fusion and synthesis of muscle proteins. The nuclei in those fibers did not synthesize DNA, nor did they undergo mitosis. This approach enabled the reproducible establishment of myogenic cell lines from very small populations of myoblasts or single primary myogenic clones. Activated p53 also readily immortalized cells in primary muscle cultures, however the cells of eight out of the nine cell lines isolated had a fibroblastic morphology and could not be induced to form multinucleated fibers.     

Engineered bromodomains to explore the acetylproteome

MS‐based analysis of the acetylproteome has highlighted a role for acetylation in a wide array of biological processes including gene regulation, metabolism, and cellular signaling. To date, anti‐acetyllysine antibodies have been used as the predominant affinity reagent for enrichment of acetyllysine‐containing peptides and proteins; however, these reagents suffer from high nonspecific binding and lot‐to‐lot variability. Bromodomains represent potential affinity reagents for acetylated proteins and peptides, given their natural role in recognition of acetylated sequence motifs in vivo. To evaluate their efficacy, we generated recombinant proteins representing all known yeast bromodomains. Bromodomain specificity for acetylated peptides was determined using degenerate peptide arrays, leading to the observation that different bromodomains display a wide array of binding specificities. Despite their relatively weak affinity, we demonstrate the ability of selected bromodomains to enrich acetylated peptides from a complex biological mixture prior to mass spectrometric analysis. Finally, we demonstrate a method for improving the utility of bromodomain enrichment for MS through engineering novel affinity reagents using combinatorial tandem bromodomain pairs.     

The Genetics of Bene Israel from India Reveals Both Substantial Jewish and Indian Ancestry

The Bene Israel Jewish community from West India is a unique population whose history before the 18^th century remains largely unknown. Bene Israel members consider themselves as descendants of Jews, yet the identity of Jewish ancestors and their arrival time to India are unknown, with speculations on arrival time varying between the 8th century BCE and the 6th century CE. Here, we characterize the genetic history of Bene Israel by collecting and genotyping 18 Bene Israel individuals. Combining with 486 individuals from 41 other Jewish, Indian and Pakistani populations, and additional individuals from worldwide populations, we conducted comprehensive genome-wide analyses based on F_ST, principal component analysis, ADMIXTURE, identity-by-descent sharing, admixture linkage disequilibrium decay, haplotype sharing and allele sharing autocorrelation decay, as well as contrasted patterns between the X chromosome and the autosomes. The genetics of Bene Israel individuals resemble local Indian populations, while at the same time constituting a clearly separated and unique population in India. They are unique among Indian and Pakistani populations we analyzed in sharing considerable genetic ancestry with other Jewish populations. Putting together the results from all analyses point to Bene Israel being an admixed population with both Jewish and Indian ancestry, with the genetic contribution of each of these ancestral populations being substantial. The admixture took place in the last millennium, about 19–33 generations ago. It involved Middle-Eastern Jews and was sex-biased, with more male Jewish and local female contribution. It was followed by a population bottleneck and high endogamy, which can lead to increased prevalence of recessive diseases in this population. This study provides an example of how genetic analysis advances our knowledge of human history in cases where other disciplines lack the relevant data to do so.     

Characterizing the Google Books Corpus: Strong Limits to Inferences of Socio-Cultural and Linguistic Evolution

It is tempting to treat frequency trends from the Google Books data sets as indicators of the “true” popularity of various words and phrases. Doing so allows us to draw quantitatively strong conclusions about the evolution of cultural perception of a given topic, such as time or gender. However, the Google Books corpus suffers from a number of limitations which make it an obscure mask of cultural popularity. A primary issue is that the corpus is in effect a library, containing one of each book. A single, prolific author is thereby able to noticeably insert new phrases into the Google Books lexicon, whether the author is widely read or not. With this understood, the Google Books corpus remains an important data set to be considered more lexicon-like than text-like. Here, we show that a distinct problematic feature arises from the inclusion of scientific texts, which have become an increasingly substantive portion of the corpus throughout the 1900s. The result is a surge of phrases typical to academic articles but less common in general, such as references to time in the form of citations. We use information theoretic methods to highlight these dynamics by examining and comparing major contributions via a divergence measure of English data sets between decades in the period 1800–2000. We find that only the English Fiction data set from the second version of the corpus is not heavily affected by professional texts. Overall, our findings call into question the vast majority of existing claims drawn from the Google Books corpus, and point to the need to fully characterize the dynamics of the corpus before using these data sets to draw broad conclusions about cultural and linguistic evolution.     

Quantitative Ultrasound Spectroscopic Imaging for Characterization of Disease Extent in Prostate Cancer Patients

Three-dimensional quantitative ultrasound spectroscopic imaging of prostate was investigated clinically for the noninvasive detection and extent characterization of disease in cancer patients and compared to whole-mount, whole-gland histopathology of radical prostatectomy specimens. Fifteen patients with prostate cancer underwent a volumetric transrectal ultrasound scan before radical prostatectomy. Conventional-frequency (~ 5 MHz) ultrasound images and radiofrequency data were collected from patients. Normalized power spectra were used as the basis of quantitative ultrasound spectroscopy. Specifically, color-coded parametric maps of 0-MHz intercept, midband fit, and spectral slope were computed and used to characterize prostate tissue in ultrasound images. Areas of cancer were identified in whole-mount histopathology specimens, and disease extent was correlated to that estimated from quantitative ultrasound parametric images. Midband fit and 0-MHz intercept parameters were found to be best associated with the presence of disease as located on histopathology whole-mount sections. Obtained results indicated a correlation between disease extent estimated noninvasively based on midband fit parametric images and that identified histopathologically on prostatectomy specimens, with an r² value of 0.71 (P < .0001). The 0-MHz intercept parameter demonstrated a lower level of correlation with histopathology. Spectral slope parametric maps offered no discrimination of disease. Multiple regression analysis produced a hybrid disease characterization model (r² = 0.764, P < .05), implying that the midband fit biomarker had the greatest correlation with the histopathologic extent of disease. This work demonstrates that quantitative ultrasound spectroscopic imaging can be used for detecting prostate cancer and characterizing disease extent noninvasively, with corresponding gross three-dimensional histopathologic correlation.     

Pancreatic polypeptide inhibits somatostatin secretion

Pancreatic polypeptide (PP) is a major agonist for neuropeptide Y4 receptors (NPY4R). While NPY4R has been identified in various tissues, the cells on which it is expressed and its function in those cells has not been clearly delineated. Here we report that NPY4R is present in all somatostatin-containing cells of tissues that we tested, including pancreatic islets, duodenum, hippocampus, and hypothalamus. Its agonism by PP decreases somatostatin secretion from human islets. Mouse embryonic hippocampal (mHippo E18) cells expressed NPY4Rs and their activation by PP consistently decreased somatostatin secretion. Furthermore, central injection of PP in mice induced c-Fos immunoreactivity in somatostatin-containing cells in the hippocampus compared with PBS-injected mice. In sum, our results identify PP as a pivotal modulator of somatostatin secretion.     

Comparative cross-species alternative splicing in plants

Alternative splicing (AS) can add significantly to genome complexity. Plants are thought to exhibit less AS than animals. An algorithm, based on expressed sequence tag (EST) pairs gapped alignment, was developed that takes advantage of the relatively small intron and exon size in plants and directly compares pairs of ESTs to search for AS. EST pairs gapped alignment was first evaluated in Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and tomato (Solanum lycopersicum) for which annotated genome sequence is available and was shown to accurately predict splicing events. The method was then applied to 11 plant species that include 17 cultivars for which enough ESTs are available. The results show a large, 3.7-fold difference in AS rates between plant species with Arabidopsis and rice in the lower range and lettuce (Lactuca sativa) and sorghum (Sorghum bicolor) in the upper range. Hence, compared to higher animals, plants show a much greater degree of variety in their AS rates and in some plant species the rates of animal and plant AS are comparable although the distribution of AS types may differ. In eudicots but not monocots, a correlation between genome size and AS rates was detected, implying that in eudicots the mechanisms that lead to larger genomes are a driving force for the evolution of AS.     

Specific targeting to murine myeloma cells of Cyt1Aa toxin from Bacillus thuringiensis subspecies israelensis

Multiple myeloma is currently an incurable cancer of plasma B cells often characterized by overproduction of abnormally high quantities of a patient-specific, clonotypic immunoglobulin "M-protein." The M-protein is expressed on the cell membrane and secreted into the blood. We previously showed that ligand-toxin conjugates (LTC) incorporating the ribosome-inactivating Ricin-A toxin were very effective in specific cytolysis of the anti-ligand antibody-bearing target cells used as models for multiple myeloma. Here, we report on the incorporation of the membrane-disruptive Cyt1Aa toxin from Bacillus thuringiensis subsp. israelensis into LTCs targeted to murine myeloma cells. Proteolytically activated Cyt1Aa was conjugated chemically or genetically through either its amino or carboxyl termini to the major peptidic epitope VHFFKNIVTPRTP (p87-99) of the myelin basic protein. The recombinant fusion-encoding genes were cloned and expressed in acrystalliferous B. thuringiensis subsp. israelensis through the shuttle vector pHT315. Both chemically conjugated and genetically fused LTCs were toxic to anti-myelin basic protein-expressing murine hybridoma cells, but the recombinant conjugates were more active. LTCs comprising the Cyt1Aa toxin might be useful anticancer agents. As a membrane-acting toxin, Cyt1Aa is not likely to induce development of resistant cell lines.     

Rhomboid cleaves Star to regulate the levels of secreted Spitz

Intracellular trafficking of the precursor of Spitz (Spi), the major Drosophila EGF receptor (EGFR) ligand, is facilitated by the chaperone Star, a type II transmembrane protein. This study identifies a novel mechanism for modulating the activity of Star, thereby influencing the levels of active Spi ligand produced. We demonstrate that Star can efficiently traffic Spi even when present at sub-stoichiometric levels, and that in Drosophila S(2)R(+) cells, Spi is trafficked from the endoplasmic reticulum to the late endosome compartment, also enriched for Rhomboid, an intramembrane protease. Rhomboid, which cleaves the Spi precursor, is now shown to also cleave Star within its transmembrane domain both in cell culture and in flies, expanding the repertoire of known Rhomboid substrates to include both type I and type II transmembrane proteins. Cleavage of Star restricts the amount of Spi that is trafficked, and may explain the exceptional dosage sensitivity of the Star locus in flies.