Direct solid phase sequencing of genomic and plasmid DNA using magnetic beads as solid support.

Approaches to direct solid phase sequencing of genomic and plasmid DNA have been developed using magnetic beads, coated with streptavidin, as solid support. The DNA is immobilized through selective incorporation of biotin into one of the strands. A single stranded template, suitable for sequencing, is obtained through strand-specific elution. Using this concept, in vitro amplified plasmid DNA and chromosomal DNA were sequenced directly from single colonies. The solid phase approach ensures that the amplification and the sequencing reactions can be performed under optimal conditions. The system was found to be suitable for sequencing using both isotope- and fluorescent-labelled primers.     

Physiological profile of world-class high-altitude climbers

The functional characteristics of six world-class high-altitude mountaineers were assessed 2-12 mo after the last high-altitude climb. Each climber on one or several occasions had reached altitudes of 8,500 m or above without supplementary O2. Static and dynamic lung volumes and right and left echocardiographic measurements were found to be within normal limits of sedentary controls (SC). Muscle fiber distribution was 70% type I, 22% type IIa, and 7% type IIb. Mean muscle fiber cross-sectional area was significantly smaller than that of SC (-15%) and of long-distance runners (LDR, -51%). The number of capillaries per unit cross-sectional area was significantly greater than that of SC (+ 40%). Total mitochondrial volume was not significantly different from that of SC, but its subsarcolemmal component was equal to that of LDR. Average maximal O2 consumption was 60 +/- 6 ml X kg-1 X min-1, which is between the values of SC and LDR. Average maximal anaerobic power was 28 +/- 2.5 W X kg-1, which is equal to that of SC and 40% lower that that of competitive high jumpers. All subjects were characterized by resting hyperventilation both in normoxia and in moderate (inspired O2 partial pressure = 77 Torr) hypoxia resulting in higher oxyhemoglobin saturation levels in hypoxia. The ventilatory response to four tidal volumes of pure O2 was similar to that of SC. It is concluded that elite high-altitude climbers do not have physiological adaptations to high altitude that justify their unique performance.     

The IsTaR 1 serodeme of Trypanosoma brucei: development of a new serodeme

An extensive serodeme of sequentially-isolated antigenic variants of African trypanosomes has been produced from both syringe-passaged and cyclically-transmitted Trypanosoma brucei of the IsTaR 1 clone derived from EATRO 164. The majority of the antigenic variants were isolated from chronically-infected deer mice (Peromyscus leucopus). The pattern of parasitemias during the course of infections initiated with syringe-passaged trypanosomes differed from those initiated with cyclically-transmitted trypanosomes. Trypanosome populations from syringe-passaged (192) and cyclically-transmitted (31) clones were each amplified by growth in lethally-irradiated mice and cryopreserved for retrospective analysis. Five clones derived from a single deer mouse during the first 44 days of infection, and 2 clones derived from an acutely-infected rat were established from these amplified populations. Homogeneous populations were grown in lethally-irradiated rats and mice for antigenic analysis purification of variant-specific glycoprotein. Six of the 7 clones were distinct variants by immunological criteria using antisera derived from whole cells or purified surface glycoproteins. Two clones, one derived from the acutely-infected rat, and the other from the first parasitemia in a chronic infection that was initiated with the former clone, were immunologically identical. Production of these clones established a well-defined serodeme that will allow detailed analysis of antigenic variation.     

Anaerobic energy release in skeletal muscle during electrical stimulation in men

The quadriceps femoris muscles of seven men were electrically stimulated under extended anaerobic conditions to quantitate anaerobic energy release and the contribution of the glycolytic system to total ATP production. Muscles were intermittently stimulated 64 times at 20 Hz while leg blood flow was occluded. Each contraction lasted 1.6 s and was followed by 1.6 s of rest. The total contraction time was 102.4 s. Muscle biopsies were taken at rest and following 16, 32, 48, and 64 contractions. The ATP turnover rates during the four 16-contraction periods were 6.12, 2.56, 2.17, and 0.64 mmol X kg dry muscle-1 X s-1 contraction time. Glycolysis provided 58%, phosphocreatine 40% and a decreased ATP store 2% of the consumed energy during the initial 16 contractions. Glycolysis was responsible for 90% of the total ATP production beyond contraction 16. Absolute glycolytic ATP production decreased to 60, 55, and 17% of the amount in the initial 16 contractions during the final three periods, respectively. In conclusion glycolysis produced approximately 195 mmol ATP/kg dry muscle during the initial 48 contractions (76.8 s) and only approximately 15 mmol ATP/kg dry muscle during the final 16 contractions. Equivalent values for total ATP turnover were 278 and 16.5 mmol/kg dry muscle.     

Trypanosome hybrids generated in tsetse flies by nuclear fusion.

Genetic exchange may occur between two particular Trypanosoma brucei clones simultaneously transmitted by the same tsetse fly. We report here that this exchange takes place in the fly, through nuclear fusion. The resulting hybrids appear to be sub-tetraploid, some particular DNA sequences from one of the parental stocks being lost before enough cloned hybrid trypanosomes could be harvested for DNA analysis. A further reduction of the DNA content of these hybrids occurs gradually upon growth and yields near diploid value in a major part of the population. This mode of hybrid generation is different from the fusion of haploid gametes, which is thought to occur normally upon inoculation of metacyclic trypanosomes in their mammalian host. In this respect, the sub-tetraploid hybrids appear to undergo meiosis in the fly, generating sub-diploid metacyclic forms, then fusion in the mammalian blood.     

Semi-automated solid-phase DNA sequencing.

Increasing the efficiency of DNA sequencing necessitates the development of systems which reduce the need for manual operations by integrating template preparation, sequencing reactions, product separation and detection. A semi-automated system, whereby PCR-amplified biotinylated genomic or plasmid DNA is immobilized on streptavidin-coated magnetic beads, has been developed.     

Defining essential stem cell characteristics in adipose-derived stromal cells extracted from distinct anatomical sites

The discovery of adipose-derived stromal cells (ASCs) has created many opportunities for the development of patient-specific cell-based replacement therapies. We have isolated multiple cell strains of ASCs from various anatomical sites (abdomen, arms/legs, breast, buttocks), indicating widespread distribution of ASCs throughout the body. Unfortunately, there exists a general lack of agreement in the literature as to their "stem cell" characteristics. We find that telomerase activity and expression of its catalytic subunit in ASCs are both below the levels of detection, independent of age and culturing conditions. ASCs also undergo telomere attrition and eventually senesce, while maintaining a stable karyotype without the development of spontaneous tumor-associated abnormalities. Using a set of cell surface markers that have been promoted to identify ASCs, we find that they failed to distinguish ASCs from normal fibroblasts, as both are positive for CD29, CD73 and CD105 and negative for CD14, CD31 and CD45. All of the ASC isolates are multipotent, capable of differentiating into osteocytes, chondrocytes and adipocytes, while fibroblasts show no differentiation potential. Our ASC strains also show elevated expression of genes associated with pluripotent cells, Oct-4, SOX2 and NANOG, when compared to fibroblasts and bone marrow-derived mesenchymal stem cells (BM-MSCs), although the levels were lower than induced pluripotent stem cells (iPS). Together, our data suggest that, while the cell surface profile of ASCs does not distinguish them from normal fibroblasts, their differentiation capacity and the expression of genes closely linked to pluripotency clearly define ASCs as multipotent stem cells, regardless of tissue isolation location.     

Bi-directional modulation of fast inhibitory synaptic transmission by leptin

The hormone leptin has widespread actions in the CNS. Indeed, leptin markedly influences hippocampal excitatory synaptic transmission and synaptic plasticity. However, the effects of leptin on fast inhibitory synaptic transmission in the hippocampus have not been evaluated. Here, we show that leptin modulates GABA_A receptor-mediated synaptic transmission onto hippocampal CA1 pyramidal cells. Leptin promotes a rapid and reversible increase in the amplitude of evoked GABA_A receptor-mediated inhibitory synaptic currents (IPSCs); an effect that was paralleled by increases in the frequency and amplitude of miniature IPSCs, but with no change in paired pulse ratio or coefficient of variation, suggesting a post-synaptic expression mechanism. Following washout of leptin, a persistent depression (inhibitory long-lasting depression) of evoked IPSCs was observed. Whole-cell dialysis or bath application of inhibitors of phosphoinositide 3 (PI 3)-kinase or Akt prevented leptin-induced enhancement of IPSCs indicating involvement of a post-synaptic PI 3-kinase/Akt-dependent pathway. In contrast, blockade of PI 3-kinase or Akt activity failed to alter the ability of leptin to induce inhibitory long-lasting depression, suggesting that this process is independent of PI 3-kinase/Akt. In conclusion these data indicate that the hormone leptin bi-directionally modulates GABA_A receptor-mediated synaptic transmission in the hippocampus. These findings have important implications for the role of this hormone in regulating hippocampal pyramidal neuron excitability.     

Leptin

It is well established that the adipocyte-derived hormone leptin is an important circulating satiety factor that regulates body weight and food intake via its actions on specific hypothalamic nuclei. However, there is growing evidence that leptin and its receptors are widely expressed throughout the brain, in regions not generally associated with energy homeostasis, such as cortex, cerebellum, brainstem, basal ganglia, and hippocampus. In this review the author discusses recent advances made in leptin neurobiology, with particular emphasis on the role of this endocrine peptide in normal and pathophysiological hippocampal function.     

Occupancy versus colonization–extinction models for projecting population trends at different spatial scales

Understanding spatiotemporal population trends and their drivers is a key aim in population ecology. We further need to be able to predict how the dynamics and sizes of populations are affected in the long term by changing landscapes and climate. However, predictions of future population trends are sensitive to a range of modeling assumptions. Deadwood‐dependent fungi are an excellent system for testing the performance of different predictive models of sessile species as these species have different rarity and spatial population dynamics, the populations are structured at different spatial scales, and they utilize distinct substrates. We tested how the projected large‐scale occupancies of species with differing landscape‐scale occupancies are affected over the coming century by different modeling assumptions. We compared projections based on occupancy models against colonization–extinction models, conducting the modeling at alternative spatial scales and using fine‐ or coarse‐resolution deadwood data. We also tested effects of key explanatory variables on species occurrence and colonization–extinction dynamics. The hierarchical Bayesian models applied were fitted to an extensive repeated survey of deadwood and fungi at 174 patches. We projected higher occurrence probabilities and more positive trends using the occupancy models compared to the colonization–extinction models, with greater difference for the species with lower occupancy, colonization rate, and colonization:extinction ratio than for the species with higher estimates of these statistics. The magnitude of future increase in occupancy depended strongly on the spatial modeling scale and resource resolution. We encourage using colonization–extinction models over occupancy models, modeling the process at the finest resource‐unit resolution that is utilizable by the species, and conducting projections for the same spatial scale and resource resolution at which the model fitting is conducted. Further, the models applied should include key variables driving the metapopulation dynamics, such as the availability of suitable resource units, habitat quality, and spatial connectivity.