Synthesis and expression of genes encoding tuna, pigeon, and horse cytochromes c in the yeast Saccharomyces cerevisiae.

Genes encoding tuna, pigeon, and horse cytochromes c were constructed with synthetic oligodeoxyribonucleotides having preferred codons and portions of the iso-1-cytochrome c-encoding gene from the yeast Saccharomyces cerevisiae. The genes were ligated into an expression vector, which contains the normal 5'- and 3'-untranslated regions of the yeast iso-1-cytochrome c gene, and were integrated in single copy into the chromosome. Yeast strains were also constructed with multiple integrated copies of the pigeon gene. The heterologous and normal mRNA levels of the single-copy strains were equivalent. Although the N-terminal methionines were completely cleaved in the heterospecific proteins, the levels of trimethylation of Lys72 and acetylation of N-terminal glycines ranged from 39-78% and 10-70%, respectively. Horse cytochrome c was produced at a nearly normal level, whereas the pigeon and tuna cytochromes c were produced at approx. 40% of the normal levels. The levels of the cytochromes c and growth of the mutant yeast strains indicated that the heterospecific cytochromes c had approx. 50% specific activity in vivo.     

Recurrent Genomic Gains in Preinvasive Lesions as a Biomarker of Risk for Lung Cancer

Lung carcinoma development is accompanied by field changes that may have diagnostic significance. We have previously shown the importance of chromosomal aneusomy in lung cancer progression. Here, we tested whether genomic gains in six specific loci, TP63 on 3q28, EGFR on 7p12, MYC on 8q24, 5p15.2, and centromeric regions for chromosomes 3 (CEP3) and 6 (CEP6), may provide further value in the prediction of lung cancer. Bronchial biopsy specimens were obtained by LIFE bronchoscopy from 70 subjects (27 with prevalent lung cancers and 43 individuals without lung cancer). Twenty six biopsies were read as moderate dysplasia, 21 as severe dysplasia and 23 as carcinoma in situ (CIS). Four-micron paraffin sections were submitted to a 4-target FISH assay (LAVysion, Abbott Molecular) and reprobed for TP63 and CEP 3 sequences. Spot counts were obtained in 30–50 nuclei per specimen for each probe. Increased gene copy number in 4 of the 6 probes was associated with increased risk of being diagnosed with lung cancer both in unadjusted analyses (odds ratio = 11, p<0.05) and adjusted for histology grade (odds ratio = 17, p<0.05). The most informative 4 probes were TP63, MYC, CEP3 and CEP6. The combination of these 4 probes offered a sensitivity of 82% for lung cancer and a specificity of 58%. These results indicate that specific cytogenetic alterations present in preinvasive lung lesions are closely associated with the diagnosis of lung cancer and may therefore have value in assessing lung cancer risk.     

Effect of Testosterone on Neuronal Morphology and Neuritic Growth of Fetal Lamb Hypothalamus-Preoptic Area and Cerebral Cortex in Primary Culture

Testosterone plays an essential role in sexual differentiation of the male sheep brain. The ovine sexually dimorphic nucleus (oSDN), is 2 to 3 times larger in males than in females, and this sex difference is under the control of testosterone. The effect of testosterone on oSDN volume may result from enhanced expansion of soma areas and/or dendritic fields. To test this hypothesis, cells derived from the hypothalamus-preoptic area (HPOA) and cerebral cortex (CTX) of lamb fetuses were grown in primary culture to examine the direct morphological effects of testosterone on these cellular components. We found that within two days of plating, neurons derived from both the HPOA and CTX extend neuritic processes and express androgen receptors and aromatase immunoreactivity. Both treated and control neurites continue to grow and branch with increasing time in culture. Treatment with testosterone (10 nM) for 3 days significantly (P < 0.05) increased both total neurite outgrowth (35%) and soma size (8%) in the HPOA and outgrowth (21%) and number of branch points (33%) in the CTX. These findings indicate that testosterone-induced somal enlargement and neurite outgrowth in fetal lamb neurons may contribute to the development of a fully masculine sheep brain.     

Development of an Accelerometer-Linked Online Intervention System to Promote Physical Activity in Adolescents

Most adolescents do not achieve the recommended levels of moderate-to-vigorous physical activity (MVPA), placing them at increased risk for a diverse array of chronic diseases in adulthood. There is a great need for scalable and effective interventions that can increase MVPA in adolescents. Here we report the results of a measurement validation study and a preliminary proof-of-concept experiment testing the impact of Zamzee, an accelerometer-linked online intervention system that combines proximal performance feedback and incentive motivation features to promote MVPA. In a calibration study that parametrically varied levels of physical activity in 31 12-14 year-old children, the Zamzee activity meter was shown to provide a valid measure of MVPA (sensitivity in detecting MVPA = 85.9%, specificity = 97.5%, and r = .94 correspondence with the benchmark RT3 accelerometer system; all p < .0001). In a subsequent randomized controlled multi-site experiment involving 182 middle school-aged children assessed for MVPA over 6 wks, intent-to-treat analyses found that those who received access to the Zamzee intervention had average MVPA levels 54% greater than those of a passive control group (p < 0.0001) and 68% greater than those of an active control group that received access to a commercially available active videogame (p < .0001). Zamzee’s effects on MVPA did not diminish significantly over the course of the 6-wk study period, and were statistically significant in both females and males, and in normal- vs. high-BMI subgroups. These results provide promising initial indications that combining the Zamzee activity meter with online proximal performance feedback and incentive motivation features can positively impact MVPA levels in adolescents.     

A recognition component of the ubiquitin system is required for peptide transport in Saccharomyces cerevisiae

Peptide transport in Saccharomyces cerevisiae is controlled by three genes: PTR1, PTR2, and PTR3. PTR1 was cloned and sequenced and found to be identical to UBR1, a gene previously described as encoding the recognition component of the N-end-rule pathway of the ubiquitin-dependent proteolytic system. Independently derived ubr1 mutants, like ptr1 mutants, were unable to transport small peptides into ceils. Concomitantly, ptr1 mutants, like ubr1 mutants, were unable to degrade an engineered substrate of the N-end-rule pathway. Further, ptr1 mutants did not express PTR2, a gene encoding the integral membrane component required for peptide transport in S. cerevisiae. These results establish a physiological role for a protein previously known to be required for the degradation of N-end-rule substrates. Our findings show that peptide transport and the ubiquitin pathway—two dynamic phenomena universal to eukaryotic cells—share a common component, namely UBR1/PTR1.     

Function of minor polypeptides in foot-and-mouth disease virus and poliovirus

Foot-and-mouth disease virus and poliovirus each contain several minor polypeptides, in addition to the four structural proteins. One of these, the viral RNA polymerase, can also act as a nuclease, hydrolysing the RNA and thus destroying viral infectivity. It is tightly bound to the RNA and may be the packaging signal for assembly of the particle.     

Topological and phenomenological classification of bursting oscillations

We describe a classification scheme for bursting oscillations which encompasses many of those found in the literature on bursting in excitable media. This is an extension of the scheme of Rinzel (inMathematical Topics in Population Biology, Springer, Berlin, 1987), put in the context of a sequence of horizontal cuts through a two-parameter bifurcation diagram. We use this to describe the phenomenological character of different types of bursting, addressing the issue of how well the bursting can be characterized given the limited amount of information often available in experimental settings.