Unbalanced expression of the different subunits of elongation factor 1 in diabetic skeletal muscle

In studies using subtraction cloning to screen for alterations in mRNA expression in skeletal muscle from humans with Type 2 diabetes mellitus and control subjects, one of the most prominent differences was in the mRNA for elongation factor (EF)-1α. With Northern blot analysis, EF-1α expression was enhanced by 2- to 6-fold in both Types 1 and 2 human diabetics. In contrast, no changes in expression of EF-1β or -γ were noted. We observed similar results in animal models of Type 1 diabetes. EF-1α expression, but not EF-1β or -γ expression, was also enhanced in streptozotocin-induced diabetic rats, and this effect was reversed by insulin treatment. An increased level of EF-1α mRNA was also observed in nonobese diabetic mice. This unbalanced regulation of the expression of the different subunits of EF-1 may contribute to alterations not only in protein synthesis but also in other cellular events observed in the diabetic state.

The Activity of Differentiation Factors Induces Apoptosis in Polyomavirus Large T-Expressing Myoblasts

It is commonly accepted that pathways that regulate proliferation/differentiation processes, if altered in their normal interplay, can lead to the induction of programmed cell death. In a previous work we reported that Polyoma virus Large Tumor antigen (PyLT) interferes with in vitro terminal differentiation of skeletal myoblasts by binding and inactivating the retinoblastoma antioncogene product. This inhibition occurs after the activation of some early steps of the myogenic program. In the present work we report that myoblasts expressing wild-type PyLT, when subjected to differentiation stimuli, undergo cell death and that this cell death can be defined as apoptosis. Apoptosis in PyLT-expressing myoblasts starts after growth factors removal, is promoted by cell confluence, and is temporally correlated with the expression of early markers of myogenic differentiation. The block of the initial events of myogenesis by transforming growth factor β or basic fibroblast growth factor prevents PyLT-induced apoptosis, while the acceleration of this process by the overexpression of the muscle-regulatory factor MyoD further increases cell death in this system. MyoD can induce PyLT-expressing myoblasts to accumulate RB, p21, and muscle- specific genes but is unable to induce G00 arrest. Several markers of different phases of the cell cycle, such as cyclin A, cdk-2, and cdc-2, fail to be down-regulated, indicating the occurrence of cell cycle progression. It has been frequently suggested that apoptosis can result from an unbalanced cell cycle progression in the presence of a contrasting signal, such as growth factor deprivation. Our data involve differentiation pathways, as a further contrasting signal, in the generation of this conflict during myoblast cell apoptosis.

Fluorescence-based directed termination PCR: direct mutation characterization without sequencing

We describe a fluorescence-based directed termination PCR (fluorescent DT–PCR) that allows accurate determination of actual sequence changes without dideoxy DNA sequencing. This is achieved using near infrared dye-labeled primers and performing two PCR reactions under low and unbalanced dNTP concentrations. Visualization of resulting termination fragments is accomplished with a dual dye Li-cor DNA sequencer. As each DT–PCR reaction generates two sets of terminating fragments, a pair of complementary reactions with limiting dATP and dCTP collectively provide information on the entire sequence of a target DNA, allowing an accurate determination of any base change. Blind analysis of 78 mutants of the supF reporter gene using fluorescent DT–PCR not only correctly determined the nature and position of all types of substitution mutations in the supF gene, but also allowed rapid scanning of the signature sequences among identical mutations. The method provides simplicity in the generation of terminating fragments and 100% accuracy in mutation characterization. Fluorescent DT–PCR was successfully used to generate a UV-induced spectrum of mutations in the supF gene following replication on a single plate of human DNA repair-deficient cells. We anticipate that the automated DT–PCR method will serve as a cost-effective alternative to dideoxy sequencing in studies involving large-scale analysis for nucleotide sequence changes.

Spectral karyotyping suggests additional subsets of colorectal cancers characterized by pattern of chromosome rearrangement

The abundant chromosome abnormalities in most carcinomas are probably a reflection of genomic instability present in the tumor, so the pattern and variability of chromosome abnormalities will reflect the mechanism of instability combined with the effects of selection. Chromosome rearrangement was investigated in 17 colorectal carcinoma-derived cell lines. Comparative genomic hybridization showed that the chromosome changes were representative of those found in primary tumors. Spectral karyotyping (SKY) showed that translocations were very varied and mostly unbalanced, with no translocation occurring in more than three lines. At least three karyotype patterns could be distinguished. Some lines had few chromosome abnormalities: they all showed microsatellite instability, the replication error (RER)+ phenotype. Most lines had many chromosome abnormalities: at least seven showed a surprisingly consistent pattern, characterized by multiple unbalanced translocations and intermetaphase variation, with chromosome numbers around triploid, 6–16 structural aberrations, and similarities in gains and losses. Almost all of these were RER−, but one, LS411, was RER+. The line HCA7 showed a novel pattern, suggesting a third kind of genomic instability: multiple reciprocal translocations, with little numerical change or variability. This line was also RER+. The coexistence in one tumor of two kinds of genomic instability is to be expected if the underlying defects are selected for in tumor evolution.

Free amino acids exhibit anthozoan "host factor" activity: they induce the release of photosynthate from symbiotic dinoflagellates in vitro.

Reef-building corals and other tropical anthozoans harbor endosymbiotic dinoflagellates. It is now recognized that the dinoflagellates are fundamental to the biology of their hosts, and their carbon and nitrogen metabolisms are linked in important ways. Unlike free living species, growth of symbiotic dinoflagellates is unbalanced and a substantial fraction of the carbon fixed daily by symbiont photosynthesis is released and used by the host for respiration and growth. Release of fixed carbon as low molecular weight compounds by freshly isolated symbiotic dinoflagellates is evoked by a factor (i.e., a chemical agent) present in a homogenate of host tissue. We have identified this "host factor" in the Hawaiian coral Pocillopora damicornis as a set of free amino acids. Synthetic amino acid mixtures, based on the measured free amino acid pools of P. damicornis tissues, not only elicit the selective release of 14C-labeled photosynthetic products from isolated symbiotic dinoflagellates but also enhance total 14CO2 fixation.