Cdc13 delivers separate complexes to the telomere for end protection and replication

In Saccharomyces cerevisiae, the telomere binding protein Cdc13 mediates telomere replication by recruiting telomerase, and also performs an essential function in chromosome end protection. We show here that delivery of the Stn1 protein to the telomere, by fusing the DNA binding domain of Cdc13 (DBD(CDC13)) to Stn1, is sufficient to rescue the lethality of a cdc13 null strain and, hence, provide end protection. Telomere replication is still defective in this strain, but can be restored by delivering telomerase to the telomere as a DBD(CDC13)-telomerase fusion. These results establish Stn1 as the primary effector of chromosome end protection, whereas the principal function of Cdc13 is to provide a loading platform to recruit complexes that provide end protection and telomere replication.     

A tale of two Cbls: interplay of c-Cbl and Cbl-b in epidermal growth factor receptor downregulation

The precise role of Cbl in epidermal growth factor (EGF) receptor (EGFR) endocytosis and trafficking remains to be fully uncovered. Here, we showed that mutant EGFR1044, which was truncated after residue 1044, did not associate with c-Cbl and was not ubiquitinated initially in response to EGF but was internalized with kinetics similar to those of wild-type EGFR. This finding indicates that c-Cbl-mediated ubiquitination is not required for EGF-induced EGFR endocytosis. We also showed that the previously identified internalization-deficient mutant receptor EGFR1010LL/AA bound to c-Cbl and was fully ubiquitinated in response to EGF, which indicates that c-Cbl binding and ubiquitination are not sufficient for EGFR internalization. We next investigated EGFR trafficking following EGFR internalization. We found that c-Cbl disassociation from EGFR occurred well in advance of EGFR degradation and that this event was concurrent with the selective dephosphorylation of EGFR at Y1045. This finding suggests that once EGFR is ubiquitinated, continual Cbl association is not required for EGFR degradation. Because EGFR1044 is ubiquitinated and degraded similarly to wild-type EGFR, we examined the role of another prominent Cbl homologue, Cbl-b, and found that Cbl-b was associated with both EGFR and EGFR1044. Further study showed that Cbl-b bound to EGFR at two regions: one in the C-terminal direction from residue 1044 and one in the N-terminal direction from residue 958. Moreover, Cbl-b association with EGFR rose markedly following a decrease in c-Cbl association, corresponding to a second peak of EGFR ubiquitination occurring later in EGFR trafficking. Using RNA interference to knock down both c-Cbl and Cbl-b, we were able to abolish EGFR downregulation. This knockdown had no affect on the rate of EGF-induced EGFR internalization. We found that the two Cbls accounted for total receptor ubiquitination and that while c-Cbl and Cbl-b are each alone sufficient to effect EGFR degradation, both are involved in the physiological, EGF-mediated process of receptor downregulation. Furthermore, these data ultimately reveal a previously unacknowledged temporal interplay of two major Cbl homologues with the trafficking of EGFR.     

Transfer of Tn1545 and Tn916 to Clostridium acetobutylicum

Tn1545, a conjugative transposon originally discovered in Streptococcus pneumoniae, has been transferred from Enterococcus faecalis and Bacillus subtilis to Clostridium acetobutylicum NCIB 8052. Transfer between different strains of C. acetobutylicum has also been observed. Insertion of Tn1545 into the C. acetobutylicum chromosome occurred at multiple sites, as shown by Southern hybridization. Although ermAM (erythromycin-resistance) was the most satisfactory marker for primary selection of transconjugants, all three Tn1545-encoded antibiotic resistance genes (aphA-3, ermAM, and tetM) were apparently expressed in C. acetobutylicum. Our results indicate that Tn1545 is potentially useful for undertaking mutagenesis and mutational cloning in this industrially important organism. Transfer of another conjugative transposon, Tn916, from E. faecalis to C. acetobutylicum NCIB 8052 was also apparently detected. Circumstantial evidence suggests that there may be a hot spot for Tn916 insertion in the C. acetobutylicum NCIB 8052 chromosome.     

Optimization of the Oxygen Transport System

Emphasizing the over-all performance of the O₂ transport system, as well as the interactions between its various subsystems, a method for a parametric performance analysis has been developed. The purpose of such an analysis is three-fold: 1. It permits an evaluation of those parameters which are critical for the performance of the system under conditions of stress. 2. It leads to an assessment of the ranking of individual members within the hierarchy of biological control systems. 3. It permits an objective assessment of the severity and prognosis of cardiovascular and respiratory diseases and of the degree of disability resulting therefrom. Starting with the principle of conservation of mass, two equations are derived which express the balance of oxygen in terms of supply, consumption, and waste. These equations are then developed in terms of the parameters of the system; namely, ventilation, inspired O₂ concentration, cardiac output, O₂ capacity of the blood, energy requirements of the two pumps, fractional extraction of O₂ from alveolar air (ventilation-perfusion ratio), and the oxygen utilization fraction in the periphery. The results indicate that the normal system attempts to maximize the oxygen utilization fraction while minimizing ventilatory and cardiac energy requirements. Changes in the ventilation-perfusion ratio are relatively less important. Possible extensions of the model are discussed.     

A microbial TLR2 agonist imparts macrophage-activating ability to apolipoprotein A-1

There is increasing epidemiologic evidence implying a role for chronic infection in atherosclerosis and that microbial TLR agonists may contribute to this disease. Mycoplasma arthritidis is an agent of acute and chronic inflammatory disease in rodents, and has been used extensively as a model for defining the mechanisms involved in arthritis and other inflammatory diseases. We have purified a 28-kDa, apolipoprotein A-1 (apoA-1)-like TLR2-dependent macrophage-activating moiety from a culture of a virulent strain of M. arthritidis. ApoA-1 similarly isolated from uninoculated mycoplasma medium was without bioactivity. The activity of the mycoplasma-derived molecule was resistant to heat and to digestion with proteinase K, but was susceptible to alkaline hydrolysis and H(2)O(2) oxidation. Infrared profiles of normal apoA-1 and that derived from mycoplasma were distinct. Unlike the activity of other mycoplasmal TLR2 agonists such as macrophage-activating lipopeptide-2, activity of the M. arthritidis-derived 28-kDa component was dependent upon CD14, a coreceptor for LPS. Finally, we showed that bioactive lipopeptides prepared from M. arthritidis grown in serum-free medium and also from a 41-kDa known bioactive lipoprotein of M. arthritidis, avidly bound to purified apoA-1 that separated out by SDS-PAGE, induced TNF-alpha and IL-12p40 both in vitro and in vivo. ApoA-1 is a key functional component of the high-density lipoprotein cholesterol complex by scavenging and removing unwanted lipids. Our finding that this molecule can acquire macrophage-activating properties from microbial TLR2-dependent agonists suggests a novel mechanism whereby some microbial agents might reverse the protective role of apoA-1, thus contributing to the genesis of atherosclerosis.     

Ontogeny tends to recapitulate phylogeny in digital organisms

Abstract Biologists have long debated whether ontogeny recapitulates phylogeny and, if so, why. Two plausible explanations are that (i) changes to early developmental stages are selected against because they tend to disrupt later development and (ii) simpler structures often precede more complex ones in both ontogeny and phylogeny if the former serve as building blocks for the latter. It is difficult to test these hypotheses experimentally in natural systems, so we used a computational system that exhibits evolutionary dynamics. We observed that ontogeny does indeed recapitulate phylogeny; traits that arose earlier in a lineage's history also tended to be expressed earlier in the development of individuals. The relative complexity of traits contributed substantially to this correlation, but a significant tendency toward recapitulation remained even after accounting for trait complexity. This additional effect provides evidence that selection against developmental disruption also contributed to the conservation of early stages in development.     

Dietary analysis of young professional soccer players for 1 week during the competitive season

Limited data exist concerning the dietary practices of young professional soccer players that compete within the United Kingdom. Therefore, the purpose of this study was to investigate the nutritional and activity habits of professional male soccer players (n = 10; age: 17 ± 1 years, height: 1.72 ± 0.01 m, mass: 67.5 ± 1.8 kg, estimated maximal aerobic capacity: 57.8 ± 0.9 ml·kg·min) who played for the youth team of a UK-based Championship club. All players recorded their 7-day dietary intake and activity habits during a competitive week that included a match day, 4- training days, and 2 rest days in the first half of the 2009/2010 playing season. The intake of carbohydrates (5.9 ± 0.4 g·kg·d), proteins (1.7 ± 0.1 g·kg·d), and fats (1.5 ± 0.1 g·kg·d) represented 56 ± 1, 16 ± 1, and 31 ± 1% of the mean daily energy intake respectively. The intake of fiber was found to be significantly lower than Recommended Nutrient Intake (RNI) values (67% of RNI, p < 0.001), whereas all other analyzed micronutrients met or exceeded recommended values. A mean daily energy deficit of 788 ± 174 kcal existed because daily energy expenditures exceeded that of intake (3,618 ± 61 vs. 2831 ± 164 kcal, p = 0.001). The mean daily fluid intake was 3.2 ± 0.3 L. Consequently, the nutritional practices of the sampled group of professional youth soccer players were inadequate to sustain optimized performance throughout training and match play. Youth soccer players should therefore seek to ensure that their diets contain adequate energy through increased total caloric intake, while also optimizing the proportion of energy derived from carbohydrates and ensuring that enough fiber-rich foods are consumed.