Carbohydrate ingestion and muscle glycogen depletion during marathon and ultramarathon racing

Two studies were undertaken to characterize the effects of carbohydrate ingestion on fuel/hormone response to exercise and muscle glycogen utilization during prolonged competitive exercise. In study 1, eighteen subjects were divided into three groups, matched for maximum oxygen consumption (VO2max) and blood lactate turnpoint. All subjects underwent a 3-day carbohydrate (CHO) depletion phase, followed by 3 days of CHO loading (500-600 g.day-1). During the race, the groups drank either 2% glucose (G), 8% glucose polymer (GP), or 8% fructose (F). Muscle biopsies were performed before and after the race and venous blood was sampled before and at regular intervals during the race. In study 2, eighteen subjects divided into 2 matched groups ingested either a 4% G or 10% GP solution during a 56 km race. Despite significantly greater CHO ingestion by GP and F in study 1 and by GP in study 2, blood glucose, free fatty acids and insulin concentrations, muscle glycogen utilization and running performance were not different between groups. These studies show (i) that hypoglycaemia is uncommon in athletes competing in races of up to 56 km provided they CHO-load before and ingest a minimum of 10 g CHO.h-1 during competition; (ii) that neither the amount (10 g vs 40 g.h-1) nor the type of carbohydrate (G vs GP vs F) has any effect on the extent of muscle glycogen depletion or running performance in matched subjects racing over distances up to 56 km.     

Studies on NK cell purification by negative selection in human peripheral blood

We have attempted to improve negative selection procedures for the large scale purification of human CD _in3 ^– CD56⁺ NK cells. In a series of experiments, purifications of NK cells from 10⁸ PBMC were performed by T cell depletion using either direct or indirect anti-CD3 labeling and the Magnetic Activated Cell Separation (MACS) procedure. Contaminating CD3⁺ cells were still present using either one of these two different T cell depletion protocols as shown by phenotyping IL-2 supplemented cell cultures on day 12. A second cycle of purification was therefore added. When MACS and Dynabeads were compared as complementary procedures to the first MACS cycle starting with 10⁸ cells, the Dynabeads method was found to be superior to the MACS with regard to the elimination of residual T cells. Starting from 10⁹ PBMC, we showed that this MACS+Dynabeads procedure gave similar satisfactory results when compared to the scaling-up of a previously established two steps procedure using Dynabeads. These two approaches (MACS+Dynabeads and 2 cycles of Dynabeads) have been also tested in a clinical setting to purify NK cells from cancer patients prior toin vitro expansion. The results indicate that the two methods are equivalent with respect to purity and recovery rate; a slight advantage in terms of feasibility was found in favor of 2 cycles of Dynabeads.     

A highly repeated DNA sequence in Arabidopsis thaliana

Summary Three members of a family of highly repeated DNA sequences from Arabidopsis thaliana have been cloned and characterized. The repeat unit has an average length of 180 bp and is tandemly repeated in arrays longer than 50 kb. This family represents more than one percent of the Arabidopsis genome. Sequence comparisons with tandemly repeated DNA sequences from other Cruciferae species show several regions of homology and a similar length of the repeat unit. Homologies are also found to highly repeated sequences from other plant species. When the sequence CCGG occurs in the repeated DNA, the inner cytosine is generally methylated.     

Cell population obtained by bronchoalveolar lavage in Pneumocystis carinii pneumonitis

In the course of bronchoalveolar lavages performed in 115 immunocompromised patients in order to investigate the occurrences of pneumonitis, Pneumocystis carinii pneumonia was diagnosed by demonstration of cysts in bronchoalveolar lavage specimens from 11 patients. The cellular phenomena associated with P. carinii infection at the level of the alveolar space were evaluated. Differential cell counts on bronchoalveolar lavage preparations stained by the May-Grünwald-Giemsa method were performed in immunocompromised patients and in ten nonimmunocompromised patients without any respiratory disease. A decrease in the alveolar macrophage count associated with an increase in the polymorphonuclear neutrophil count and the presence of plasma cells and/or immunoblasts was highly suggestive of P. carinii pneumonia. These cellular changes in bronchoalveolar lavage specimens are discussed in relation to the pathologic features usually described in P. carinii pneumonia.     

The aux1 Mutation of Arabidopsis Confers Both Auxin and Ethylene Resistance

Mutagenized populations of Arabidopsis thaliana seedlings were screened for plants capable of root growth on inhibitory concentrations of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid. Four of the mutant lines recovered from this screen display a defect in root gravitropism as well as hormone resistance. The aerial portions of these plants are similar to wild-type in appearance. Genetic analysis of these four mutants demonstrated that hormone resistance segregated as a recessive trait and that all four mutations were alleles of the auxin-resistant mutation aux1 [Maher HP, Martindale SJB (1980) Biochem Genet 18: 1041-1053]. These new mutants have been designated aux1-7, 1-12, 1-15, and 1-19. The sensitivity of wild-type and aux1-7 roots to indole-3-acetic acid, 2,4-dichlorophenoxyacetic acid, and ethylene was determined. The results of these assays show that aux1-7 plants require a 12-fold (indole-3-acetic acid) or 18-fold (2,4-dichlorophenoxyacetic acid) higher concentration of auxin than wild-type for a 50% inhibition of root growth. In addition, ethylene inhibition of root growth in aux1-7 plants is approximately 30% that of wild-type at saturating ethylene concentrations. These results indicate that aux1 plants are resistant to both auxin and ethylene. We have also determined the effect of ethylene treatment on chlorophyll loss and peroxidase activity in the leaves of aux1 and wild-type plants. No difference between mutant and wild-type plants was observed in these experiments, indicating that hormone resistance in aux1 plants may be limited to root growth. Our studies suggest that the AUX1 gene may have a specific function in the hormonal regulation of gravitropism.     

Induction of apoptosis by protein kinase C pseudosubstrate lipopeptides in several human cells

Intracellular enzymes or receptors are interesting targets for thepharmacomodulation of cellular metabolism. We have previously shown thatmodification of relatively long peptides by a palmitoyl-lysine residue couldfacilitate their delivery into the cytoplasm of living cells. Severalpeptides containing pseudosubstrate sequences of protein kinase C (PKC) havebeen evaluated for their ability to modulate phosphorylation of modelsubstrate, neuronal morphology or tumor necrosis factor secretion. In thiswork we have evaluated the effect of palmitoyl-modified PKC-pseudosubstratepeptides on induction of apoptosis. We have established that these peptidesare able to induce apoptosis in different human cell types (primaryfibroblasts, T- and B-lymphocyte cell lines) as assessed by (terminal deoxynucleotidyl transferase dUTP nick-end labelling) and DNAfragmentation. In contrast, control peptides (non-lipidicPKC-pseudosubstrate peptides and irrelevant lipopeptides) had no or littleeffect on programmed cell death. This work highlights the pharmacologicalinterest of lipopeptides and argues in favor of the potential role of PKC(s)in the cell death machinery.     

Correlation between temperature range of growth and structural transitions in membranes and lipids of Escherichia coli K12

Purified cytoplasmic and outer membranes isolated from cells of wild-type Escherichia coli grown at different temperatures were labelled with 1,6-diphenyl-1,3,5-hexatriene and anlyzed using fluorescence polarization techniques. Lipids extracted from the membranes were similarly analyzed using fluorescence polarization. The thermotropic structural transition in outer membranes changed as a function of growth temperature. The structural transition in cytoplasmic membranes and lipids extracted from either cytoplasmic or outer membranes did not change with growth temperature. These data suggest that adaptive changes which occur in the outer membrane determine the temperature range of growth of E. coli. These changes apparently require alterations in outer membrane components other than phospholipids.     

Hormone-resistant mutants of Arabidopsis have an attenuated response to agrobacterium strains

We have examined the response of the hormone-resistant mutants axr1 and axr2 of Arabidopsis thaliana to inoculation by Agrobacterium tumefaciens and Agrobacterium rhizogenes. Our results indicate that recessive mutations in the axr1 gene affect the frequency of tumor formation after inoculation with either Agrobacterium strain. In addition, tumors produced on axr1 plants were smaller than those growing on wild-type plants. These results indicate that the product of the AXR1 gene is important for both crown gall and hairy root tumor formation. In contrast, the dominant axr2 mutation has a more severe effect on the development of crown gall tumors than on hairy root tumors. Crown gall tumors produced on axr2 plants had a different morphology than wild-type tumors and did not grow when they were removed from the explant. In contrast, a large number of hairy root tumors were produced on wild-type and axr2 plants, and both types of tumors grew when they were removed from the explant. Like the roots of axr2 plants, roots produced on axr2 explants lacked root hairs.