Comparison of incremental and steady state tests of endurance training

To compare the results obtained by incremental or constant work load exercises in the evaluation of endurance conditioning, a 20-week training programme was performed by 9 healthy human subjects on the bicycle ergometer for 1 h a day, 4 days a week, at 70-80% VO2max. Before and at the end of the training programme, (1) the blood lactate response to a progressive incremental exercise (18 W increments every 2nd min until exhaustion) was used to determine the aerobic and anaerobic thresholds (AeT and AnT respectively). On a different day, (2) blood lactate concentrations were measured during two sessions of constant work load exercises of 20 min duration corresponding to the relative intensities of AeT (1st session) and AnT (2nd session) levels obtained before training. A muscle biopsy was obtained from vastus lateralis at the end of these sessions to determine muscle lactate. AeT and AnT, when expressed as % VO2max, increased with training by 17% (p less than 0.01) and 9% (p less than 0.05) respectively. Constant workload exercise performed at AeT intensity was linked before training (60% VO2max) to a blood lactate steady state (4.8 +/- 1.4 mmol.l-1) whereas, after training, AeT intensity (73% VO2max) led to a blood lactate accumulation of up to 6.6 +/- 1.7 mmol.l-1 without significant modification of muscle lactate (7.6 +/- 3.1 and 8.2 +/- 2.8 mmol.kg-1 wet weight respectively). It is concluded that increase in AeT with training may reflect transient changes linked to lower early blood lactate accumulation during incremental exercise.(ABSTRACT TRUNCATED AT 250 WORDS)     

HU and IHF, two homologous histone-like proteins of Escherichia coli, form different protein-DNA complexes with short DNA fragments.

Using the gel retardation technique we have studied the protein-DNA complexes formed between HU--the major histone-like protein of Escherichia coli--and short DNA fragments. We show that several HU heterodimers bind DNA in a regularly spaced fashion with each heterodimer occupying about 9 base pairs. The alpha 2 and beta 2 HU homodimers form the same structure as the alpha beta heterodimer on double stranded DNA. However when compared to the heterodimer, they bind single stranded DNA with higher affinity. We also show that HU and the Integration Host Factor of E. coli (IHF) form different structures with the same DNA fragments. Moreover, HU seems to enhance the DNA-binding capacity of IHF to a DNA fragment which does not contain its consensus sequence.     

Modulation of IL-4-induced human IgE production in vitro by IFN-gamma and IL-5: the role of soluble CD23 (s-CD23)

IL-4 specifically induced IgE production by peripheral blood lymphocytes or by tonsil or spleen cells from healthy donors. IL-4-induced IgE synthesis was dependent on CD4+ T cells and monocytes and was blocked by IFN-gamma, IFN-alpha, and prostaglandin E-2 (PGE-2). These substances also inhibited IL-4-induced CD23 expression and subsequent release of soluble CD23 (s-CD23). In addition, IgE production was blocked by F(ab')2 fragments of an mAb against CD23. In contrast, IL-5 enhanced IL-4-induced IgE production, provided IL-4 was added at nonsaturating concentrations. This increase in IgE production correlated quantitatively with an enhanced release of s-CD23. Collectively, these results indicate that there is a correlation between s-CD23 release and IgE production. However, s-CD23 fractionated from supernatants of the lymphoblastoid cell line RPMI-8866 was ineffective in inducing IgE production in the absence of IL-4, but acted synergistically with suboptimal concentrations of IL-4. In addition, it is demonstrated that alloreactive T-cell clones produced varying concentrations of IL-4, IL-2, or IFN-gamma upon stimulation. Only supernatants of 2/4 of these T-cell clones induced a low degree of IgE synthesis, but in the presence of anti-IFN-gamma antibodies, all four supernatants induced a strong induction of IgE production. This IgE synthesis was blocked specifically by anti-IL-4 antibodies, indicating that IL-4 is the sole inducer of IgE synthesis. Our findings demonstrate that IL-4-induced IgE production involves complex interactions of T cells, B cells, and monocytes and is positively modulated by IL-5 and s-CD23 but down-regulated by IFN-gamma, IFN-alpha, and PGE-2, respectively.     

Cloning by functional complementation, and inactivation, of theSchizosaccharomyces pombe homologue of theSaccharomyces cerevisiae geneABC1

TheSaccharomyces cerevisiae geneABC1 is required for the correct functioning of thebc ₁ complex of the mitochondrial respiratory chain. By functional complementation of aS. cerevisiae abc1 ^– mutant, we have cloned aSchizosaccharomyces pombe cDNA, whose predicted product is 50% identical to the Abc1 protein. Significant homology is also observed with bacterial, nematode, and even human amino acid sequences of unknown function, suggesting that the Abc1 protein is conserved through evolution. The cloned cDNA corresponds to a singleS. pombe geneabc1Sp, located on chromosome II, expression of which is not regulated by the carbon source. Inactivation of theabc1Sp gene by homologous gene replacement causes a respiratory deficiency which is efficiently rescued by the expression of theS. cerevisiae ABC1 gene. The inactivated strain shows a drastic decrease in thebc ₁ complex activity, a decrease in cytochromeaa3 and a slow growth phenotype. To our knowledge, this is the first example of the inactivation of a respiratory gene inS. pombe. Our results highlight the fact thatS. pombe growth is highly dependent upon respiration, and thatS. pombe could represent a valuable model for studying nucleo-mitochondrial interactions in higher eukaryotes.     

Yeast proteins can activate expression through regulatory sequences of the amdS gene of Aspergillus nidulans

The upstream regulatory region of the amdS gene of Aspergillus nidulans contains a CCAAT sequence known to be important in setting both basal and derepressed levels of expression. We have investigated whether the CCAAT-binding HAP2/3/4 complex of the yeast Saccharomyces cerevisiae can recognise this sequence in an amdS context. Sequences from the 5′ region of amdS were cloned in front of the CYCI-lacZ fusion gene bearing a minimal promoter and transformed into wild-type and hap2 strains of yeast. This study has indicated that amdS sequences are capable of promoting regulated expression of the fusion gene in response to carbon limitation. The yeast HAP2/3/4 complex can recognise the amdS CCAAT sequence and activate expression from this sequence. In addition, the results indicate that other yeast proteins can also regulate expression from the A. nidulans amdS 5′ sequences under carbon-limiting conditions.     

Deletion of the Leader Peptide of the Mitochondrially Encoded Precursor of Saccharomyces Cerevisiae Cytochrome C Oxidase Subunit II

Cytochrome c oxidase subunit II (Cox2p) of Saccharomyces cerevisiae is synthesized within mitochondria as a precursor, pre-Cox2p. The 15-amino acid leader peptide is processed after export to the intermembrane space. Leader peptides are relatively unusual in mitochondrially coded proteins: indeed mammalian Cox2p lacks a leader peptide. We generated two deletions in the S. cerevisiae COX2 gene, removing either the leader peptide (cox2-20) or the leader peptide and processing site (cox2-21) without altering either the promoter or the mRNA-specific translational activation site. When inserted into mtDNA, both deletions substantially reduced the steady-state levels of Cox2p and caused a tight nonrespiratory phenotype. A respiring pseudorevertant of the cox2-20 mutant was heteroplasmic for the original mutant mtDNA and a ρ(-) mtDNA whose deletion fused the first 251 codons of the mitochondrial gene encoding cytochrome b to the cox2-20 sequence. The resulting fusion protein was processed to yield functional Cox2p. Thus, the presence of amino-terminal cytochrome b sequence bypassed the need for the pre-Cox2p leader peptide. We propose that the pre-Cox2p leader peptide contains a targeting signal necessary for membrane insertion, without which it remains in the matrix and is rapidly degraded.     

Modeling of adaptations to physical training by using a recursive least squares algorithm

Busso, Thierry, Christian Denis, Régis Bonnefoy,André Geyssant, and Jean-René Lacour. Modeling ofadaptations to physical training by using a recursive least squaresalgorithm. J. Appl. Physiol. 82(5):1685-1693, 1997.The present study assesses the usefulnessof a systems model with time-varying parameters for describing theresponses of physical performance to training. Data for two subjectswho undertook a 14-wk training on a cycle ergometer were used to testthe proposed model, and the results were compared with a model withtime-invariant parameters. Two 4-wk periods of intensive training wereseparated by a 2-wk period of reduced training and followed by a 4-wkperiod of reduced training. The systems input ascribed to the trainingdoses was made up of interval exercises and computed in arbitraryunits. The systems output was evaluated one to five times per week byusing the endurance time at a constant workload. The time-invariantparameters were fitted from actual performances by using the leastsquares method. The time-varying parameters were fitted by using arecursive least squares algorithm. The coefficients of determinationr² were 0.875 and0.879 for the two subjects using the time-varying model, higher thanthe values of 0.682 and 0.666, respectively, obtained with thetime-invariant model. The variations over time in the model parametersresulting from the expected reduction in the residuals appearedgenerally to account for changes in responses to training. Such a modelwould be useful for investigating the underlying mechanisms ofadaptation and fatigue.

     

Interactions between peptidyl tRNA hydrolase homologs and the ribosomal release factor Mrf1 in S. pombe mitochondria

Mitochondrial translation synthesizes key subunits of the respiratory complexes. In Schizosaccharomyces pombe, strains lacking Mrf1, the mitochondrial stop codon recognition factor, are viable, suggesting that other factors can play a role in translation termination. S. pombe contains four predicted peptidyl tRNA hydrolases, two of which (Pth3 and Pth4), have a GGQ motif that is conserved in class I release factors. We show that high dosage of Pth4 can compensate for the absence of Mrf1 and loss of Pth4 exacerbates the lack of Mrf1. Also Pth4 is a component of the mitochondrial ribosome, suggesting that it could help recycling stalled ribosomes.