Unbalanced ribosome assembly in Saccharomyces cerevisiae expressing mutant 5 S rRNAs.

Mutant 5 S rRNA genes were expressed in Saccharomyces cerevisiae to further define the function of the ribosomal 5 S RNA. RNA synthesis and utilization were assayed using previously constructed markers which have been shown to be functionally neutral and easily detected by gel electrophoresis. Most mutations were found not to affect the growth rate because they were poorly expressed or could be accommodated effectively in the ribosomal structure. Two of the mutants, Y5A99U56U57 and Y5U90i5 adversely affected cell growth as well as protein synthesis in vitro. Polyribosome profiles in both of these mutants were substantially shorter, and an analysis of the ribosomal subunit composition revealed a significant imbalance with a 25-35% excess in 40 S subunits. Kinetic analyses of RNA labeling indicated very low cellular levels of mutant RNA either because it was poorly expressed (Y5U90i5) or rapidly degraded before being incorporated into mature 60 subunits (Y5A99U56U57). The results suggest that the 5 S RNA is required for the assembly of stable ribosomal 60 S subunits and raise the possibility that this RNA or, more likely, its corresponding ribonucleoprotein complex is critical for subunit assembly or even RNA processing.     

A novel regulatory role of TRAPPC9 in L-plastin-mediated osteoclast actin ring formation

Trafficking protein particle complex 9 (TRAPPC9) is a major subunit of the TRAPPII complex. TRAPPC9 has been reported to bind nuclear factor κB kinase subunit β (IKKβ) and NF-kB-inducing kinase (NIK) where it plays a role in the canonical and noncanonical of nuclear factor-κB (NF-kB) signaling pathways, receptively. The role of TRAPPC9 in protein trafficking and cytoskeleton organization in osteoclast (OC) has not been studied yet. In this study, we examined the mRNA expression of TRAPPC9 during OC differentiation. Next, we examined the colocalization of TRAPPC9 with cathepsin-K, known to mediate OC resorption suggesting that TRAPPC9 mediates the trafficking pathway within OC. To identify TRAPPC9 protein partners important for OC-mediated cytoskeleton re-organization, we conducted immunoprecipitation of TRAPPC9 in mature OCs followed by mass spectrometry analysis. Our data showed that TRAPPC9 binds various protein partners. One protein with high recovery rate is L-plastin (LPL). LPL localizes at the podosomes and reported to play a crucial role in actin aggregation thereby actin ring formation and OC function. Although the role of LPL in OC-mediated bone resorption has not fully reported in detail. Here, first, we confirmed the binding of LPL to TRAPPC9 and, then, we investigated the potential regulatory role of TRAPPC9 in LPL-mediated OC cytoskeleton reorganization. We assessed the localization of TRAPPC9 and LPL in OC and found that TRAPPC9 is colocalized with LPL at the periphery of OC. Next, we determined the effect of TRAPPC9 overexpression on LPL recruitment to the actin ring using a viral system. Interestingly, our data showed that TRAPPC9 overexpression promotes the recruitment of LPL to the actin ring when compared with control cultures. In addition, we observed that TRAPPC9 overexpression reorganizes actin clusters/aggregates and regulates vinculin recruitment into the OC periphery to initiate podosome formation.     

Evolutionary changes in the higher order structure of the ribosomal 5S RNA.

Comparative studies have been undertaken on the higher order structure of ribosomal 5S RNAs from diverse origins. Competitive reassociation studies show that 5S RNA from either a eukaryote or archaebacterium will form a stable ribonucleoprotein complex with the yeast ribosomal 5S RNA binding protein (YL3); in contrast, eubacterial RNAs will not compete in a similar fashion. Partial S1 ribonuclease digestion and ethylnitrosourea reactivity were used to probe the structural differences suggested by the reconstitution experiments. The results indicate a more compact higher order structure in eukaryotic 5S RNAs as compared to eubacteria and suggest that the archaebacterial 5S RNA contains features which are common to either group. The potential significance of these results with respect to a generalized model for the tertiary structure of the ribosomal 5S RNA and to the heterogeneity in the protein components of 5S RNA-protein complexes are discussed.     

Dynamics of changes in the cardiovascular response to submaximal exercise during low-intensity endurance training with particular reference to the systolic time intervals

Eighteen male volunteers (aged 20-23 years), not involved in any sporting activities, were submitted to 13 weeks of training consisting of 30 min exercise [at 50%-75% maximal oxygen intake (VO2max)] on a cycle ergometer, performed 3 times a week. Every 4 weeks cardiac function was evaluated by measuring the systolic time intervals at rest and during submaximal cycle exercise. Stroke volume (SV), heart rate (HR) and blood pressure (BP) responses to submaximal exercise, VO2max and anaerobic threshold (AT) were also determined. Significant increases in VO2max, increases in AT and SV at the submaximal exercise intensities, as well as decreases in HR and BP were found after 4 weeks of training. Resting systolic time intervals were not affected by training, but during the submaximal cycle exercise the values of the pre-ejection period (PEP) and isovolumic contraction time (ICT) corresponding to HR of 100 beats.min-1 were significantly lowered after 13 weeks of training, whereas PEP, ICT and total electromechanical systole corresponding to HR of 130 beats.min-1 were significantly shortened by the 4th week. The ratios of PEP:LVET (left ventricular ejection time) and ICT:LVET during submaximal exercise were significantly lowered by training starting from the 8th week. These changes might be interpreted as evidence of the training-induced enhancement of the "contractility reserve", i.e. the ability to increase heart muscle contractility with increasing exercise intensity.     

Termination region in rRNA genes from a eucaryotic thermophile, Thermomyces lanuginosus.

S1 mapping of the termination region in the ribosomal DNA from a thermophilic fungus, Thermomyces lanuginosus, revealed three distinct termini corresponding to the mature 25S rRNA, a precursor that is 19 nucleotides longer and corresponds to the 37S precursor in yeast cells, and a putative termination site at +96 that bears a limited sequence homology with the SalI box of mammalian cells. An estimate of the secondary structure suggested that the three termini are in close proximity, a feature that may be essential to precursor termination and maturation. The results raise questions regarding recently reported relationships between ribosomal DNA termination and spacer enhancer elements in fungi.     

A conserved core structure in the 18–25S rRNA intergenic region from tobacco,Nicotiana rustica

To identify conserved and functionally important features in the intergenic sequences of ribosomal DNAs, the nucleotide sequence of the 18–25S rRNA intergene region in tobacco rDNA was determined and compared to that of other higher plants. Unlike previous comparisons of more diverse organisms, sufficient sequence homology is retained in the higher plants to examine the evolutionary changes which make these regions diverse. Estimates of the secondary structure permit the identification of a core-like structure which appears to maintain the processed sites in close proximity and can be identified in the more divergent sequences.     

Exercise hyperthermia as a factor limiting physical performance: temperature effect on muscle metabolism

The muscle contents of high-energy phosphates and their derivatives [ATP, ADP, AMP, creatine phosphate (CrP), and creatine], glycogen, some glycolytic intermediates, pyruvate, and lactate were compared in 11 dogs performing prolonged heavy exercise until exhaustion (at ambient temperature 20.0 +/- 1.0 degrees C) without and with trunk cooling using ice packs. Without cooling, dogs were able to run for 57 +/- 8 min, and their rectal (Tre) and muscle (Tm) temperatures increased to 41.8 +/- 0.2 and 43.0 +/- 0.2 degrees C, respectively. Compared with noncooling, duration of exercise with cooling was longer by approximately 45% while Tre and Tm at the time corresponding to the end of exercise without cooling were lower by 1.1 +/- 0.2 and 1.2 +/- 0.2 degrees C, respectively. The muscle contents of high-energy phosphates (ATP + CrP) decreased less, the rate of glycogen depletion was lower, and the increases in the contents of AMP, pyruvate, and lactate as well as in the muscle-to-blood lactate ratio were smaller. The muscle content of lactate was positively correlated with Tm. The data indicate that with higher body temperature equilibrium between high-energy phosphate breakdown and resynthesis was shifted to the lower values of ATP and CrP and glycolysis was accelerated. The results suggest that hyperthermia developing during prolonged muscular work exerts an adverse effect on muscle metabolism that may be relevant to limitation of endurance.     

Structure of the ribosome-associated 5.8 S ribosomal RNA

The structure of the 5.8 S ribosomal RNA in rat liver ribosomes was probed by comparing dimethyl sulfate-reactive sites in whole ribosomes, 60 S subunits, the 5.8 S-28 S rRNA complex and the free 5.8 S rRNA under conditions of salt and temperature that permit protein synthesis in vitro. Differences in reactive sites between the free and both the 28 S rRNA and 60 S subunit-associated 5.8 S rRNA show that significant conformational changes occur when the molecule interacts with its cognate 28 S rRNA and as the complex is further integrated into the ribosomal structure. These results indicate that, as previously suggested by phylogenetic comparisons of the secondary structure, only the "G + C-rich" stem may remain unaltered and a universal structure is probably present only in the whole ribosome or 60 S subunit. Further comparisons with the ribosome-associated molecule indicate that while the 5.8 S rRNA may be partly localized in the ribosomal interface, four cytidylic acid residues, C56, C100, C127 and C128, remain reactive even in whole ribosomes. In contrast, the cytidylic acid residues in the 5 S rRNA are not accessible in either the 60 S subunit or the intact ribosome. The nature of the structural rearrangements and potential sites of interaction with the 28 S rRNA and ribosomal proteins are discussed.     

Greater serum GH response to arm than to leg exercise performed at equivalent oxygen uptake

The aim of this study was to provide information concerning the mechanism of exercise-induced stimulation of growth hormone (GH) release in human subjects. For this reason serum GH as well as some hemodynamic variables and blood concentrations of noradrenaline (NA), insulin (IRI), lactate (LA), glucose (BG), and free fatty acids (FFA) were determined in seven healthy male subjects exercising on a bicycle ergometer with arms or legs and running on a treadmill at equivalent oxygen consumption levels. Significantly greater increases in serum GH concentration accompanied arm exercises than those observed during the leg exercises. This was accompanied by greater increases in heart rate, blood pressure, and plasma NA and blood lactate concentrations. Serum IRI decreased during both leg exercises and did not change during the arm exercise. There were no differences in BG and plasma FFA concentrations between the three types of exercise. The role of humoral and neural signals responsible for the greater GH response to arm exercise is discussed. The findings are consistent with the hypothesis that neural afferent signals sent by muscle "metabolic receptors" participate in the activation of GH release during physical exercise. It seems likely that the stimulation of these chemoreceptors is more pronounced when smaller muscle groups are engaged at a given work load. However, a contribution of efferent impulses derived from the brain motor centres to the control system of GH secretion during exercise is also possible.