Quantitative gene expression analysis in a brain slice model: influence of temperature and incubation media

We describe the RNA integrity (28S/18S ratio) and the messenger RNA (mRNA) expression of genes encoding glyceraldehyde 3-phosphate dehydrogenase (GAPDH), microtubule-associated serine/threonine kinase 2 (Mast2), and β-actin in cortical brain slices incubated for up to 24 h in Ringer’s solution and Dulbecco’s modified Eagle’s medium (DMEM) at 25 and 37 °C. Our data reveal an optimal temporal working window between 1 and 6 h when slices are incubated in Ringer’s solution at 25 °C that allows experiments related to gene expression dynamics to be performed more suitably than those carried out at 37 °C. In addition, we show that reference gene expression may be modified in dynamic experiments and may compromise studies of gene expression.     

Sinusoidal 50 Hz magnetic fields do not affect structural morphology and proliferation of human cells in vitro

The effects of electromagnetic fields on several processes related to cell physiology and proliferation are currently being investigated. Although the results are still not conclusive and even conflicting, there seems to be a fairly good agreement on the early effects of electromagnetic fields on the generation of free radicals and on Ca++-intracellular concentration and transport. To evaluate the long-lasting consequences of these precocious events, we examined the effects of short- and long-term magnetic field exposure on structural organization (cytokeratin or actin detection), proliferation (bromodeoxyuridine incorporation and propidium iodide staining), colony forming ability and viability (trypan blue exclusion test) of highly proliferating MCF-7 cells (from human breast carcinoma) and on slowly proliferating normal human fibroblasts (from healthy donors). Cells were exposed to either 20 or 500 microT sinusoidally oscillating (50Hz) magnetic fields for different lengths of time (1 to 4 days). Short (1 day)- and long (4 days)-time exposure to the two intensities did not affect MCF-7 growth and viability, colony number and size, or cellular distribution along the cell cycle; neither were the cell morphology and the intracellular distribution and amount of cytokeratin modified. Similarly, no modifications in the actin distribution and proliferative potential were observed in normal human fibroblasts. These findings suggest that under our experimental conditions, continuous exposure to magnetic fields does not result in any appreciable effect in both normal and tumor cells in vitro.     

Dynamin participates in focal extracellular matrix degradation by invasive cells

The degradation of extracellular matrix (ECM) by matrix metalloproteases is crucial in physiological and pathological cell invasion alike. Degradation occurs at specific sites where invasive cells make contact with the ECM via specialized plasma membrane protrusions termed invadopodia. Herein, we show that the dynamin 2 (Dyn2), a GTPase implicated in the control of actin-driven cytoskeletal remodeling events and membrane transport, is necessary for focalized matrix degradation at invadopodia. Dynamin was inhibited by using two approaches: 1) expression of dominant negative GTPase-impaired or proline-rich domain-deleted Dyn2 mutants; and 2) inhibition of the dynamin regulator calcineurin by cyclosporin A. In both cases, the number and extension of ECM degradation foci were drastically reduced. To understand the site and mechanism of dynamin action, the cellular structures devoted to ECM degradation were analyzed by correlative confocal light-electron microscopy. Invadopodia were found to be organized into a previously undescribed ECM-degradation structure consisting of a large invagination of the ventral plasma membrane surface in close spatial relationship with the Golgi complex. Dyn2 seemed to be concentrated at invadopodia.     

Human cytochrome c enters murine J774 cells and causes G1 and G2/M cell cycle arrest and induction of apoptosis

Cytochrome c is well known as a carrier of electrons during respiration. Current evidence indicates that cytochrome c also functions as a major component of apoptosomes to induce apoptosis in eukaryotic cells as well as an antioxidant. More recently, a prokaryotic cytochrome c, cytochrome c(551) from Pseudomonas aeruginosa, has been shown to enter in mammalian cells such as the murine macrophage-like J774 cells and causes inhibition of cell cycle progression. Much less is known about such functions by mammalian cytochromes c, particularly the human cytochrome c. We now report that similar to P. aeruginosa cytochrome c(551), the purified human cytochrome c protein can enter J774 cells and induce cell cycle arrest at the G(1) to S phase, as well as at the G(2)/M phase at higher concentrations. Unlike P. aeruginosa cytochrome c(551) which had no effect on the induction of apoptosis, human cytochrome c induces significant apoptosis and cell death in J774 cells, presumably through inhibition of the cell cycle at the G(2)/M phase. When incubated with human breast cancer MCF-7 and normal mammary epithelial cell line MCF-10A1 cells, human cytochrome c entered in both types of cells but induced cell death only in the normal MCF-10A1 cells. The ability of human cytochrome c to enter J774 cells was greatly reduced at 4 degrees C, suggesting energy requirement in the entry process.     

Development of neural mechanisms for machine learning

The goal of this work is to develop a humanoid robot's perceptual mechanisms through the use of learning aids. We describe methods to enable learning on a humanoid robot using learning aids such as books, drawing materials, boards, educational videos or other children toys. Visual properties of objects are learned and inserted into a recognition scheme, which is then applied to acquire new object representations - we propose learning through developmental stages. Inspired in infant development, we will also boost the robot's perceptual capabilities by having a human caregiver performing educational and play activities with the robot (such as drawing, painting or playing with a toy train on a railway). We describe original algorithms to extract meaningful percepts from such learning experiments. Experimental evaluation of the algorithms corroborates the theoretical framework.     

Community ecology of small mammal populations in Panamá following an outbreak of Hantavirus pulmonary syndrome.

In late 1999 and early 2000, an outbreak of hantavirus pulmonary syndrome (HPS) occurred in and around Los Santos, on the Azuero Peninsula of southwestern Panamá. This HPS episode, resulting in 22% case fatality, was linked to the Costa Rican pigmy rice rat, Oligoryzomys fulvescens costaricensis, which harbored a then undescribed hantavirus, Choclo virus. In addition, Cherrie's cane rat, Zygodontomys brevicauda cherriei, was identified as carrying a distinct hantavirus, Calabazo virus with no known pathogenicity to humans. Herein we present the ecological results of the outbreak investigations in the Azuero region. A total of 164 animals were captured, of which 126 were potential small, non-volant mammal hosts of a hantavirus: rodents in the family Muridae. There were significant differences in small mammal community structure between case sites and a negative control site. Differences were manifest in ecological measures of species diversity and in species evenness and heterogeneity measures, as indicated by Pairwise Euclidian distances and Morisita indices of community similarity. Our analyses suggest that human activities (i.e., deforestation for cattle ranching) coupled with environmental factors (i.e., increased precipitation) may have synergistically coalesced for an increased risk of HPS to area residents.     

A comparative study of pollen trends in Genoa and Sanremo (Italy) from 1981 to 1989

Summary Trends of the atmospheric dispersion of the pollens of Urticaceae, Poaceae, Asteraceae,Plantago, Chenopodiaceae-Amaranthaceae, Polygonaceae, Oleaceae, Betulaceae-Corylaceae,Quercus, Castanea, and Cupressaceae-Taxaceae were studied from 1981 to 1989 in two localities (Genoa and Sanremo) about 120 km apart on the coast of Liguria in Italy. Statistical analysis revealed, in particular, and overall increase in pollen rain in Genoa during the period considered. The following observations were carried out on the pollens of greatest allergological interest: Urticaceae, a slightly increasing trend in Genoa; Poaceae, a moderately increasing trend in Sanremo; Oleaceae, almost parallel, uniform trendsin both centres. The values of this study lies in its ability to point out any increase which may occur over the years in the dispersal of certain allergenic pollens.     

Influence of motor units recruitment and firing rate on the soundmyogram and EMG characteristics in cat gastrocnemius

The separate contributions of the recruitment level and of the firing rate of the motor units on the soundmyogram and electromyogram time domain parameters were investigated during stimulation of the motor nerve of the cat gastrocnemius muscle. Upon orderly increase in the number of active motor units at a fixed firing rate, both the peak to peak amplitude (P-P_max) and the root mean square (RMS) of the sound myogram increased. At full recruitment the increase in firing rate from 2.5 to 50 Hz induced an exponential decline in the P-P_max. The RMS, however, followed this trend only from 15 to 50 Hz while showing an increase from 2.5 to 10 Hz. During simultaneous changes of recruitment and firing rate, the effect of increasing the number of motor units on the P-P_max and RMS is dampened by the increasing firing rate. The peak to peak amplitude of the EMG compound action potential increased with the number of active motor units. Moreover, its amplitude was not influenced by the firing rate. The EMG RMS, however, increases as a function of the firing rate. The results indicate that both the number and the firing rate of the active motor units contribute to the determination of the soundmyogram characteristics. Moreover, the peculiar changes of the soundmyogram time domain properties, compared to the ones of the EMG, allow one to differentiate the influence of the motor units number and firing rate on the electrical and mechanical performance of the muscle when stimulated.     

The influence of exercise intensity on the power spectrum of heart rate variability

The power spectral analysis of R-R interval variability (RRV) has been estimated by means of an autoregressive method in seven sedentary males at rest, during steady-state cycle exercise at 21 percent maximal oxygen uptake (%VO2max), SEM 2%, 49% VO2max, SEM 2% and 70% VO2max, SEM 2% and during recovery. The RRV, i.e. the absolute power of the spectrum, decreased 10, 100 and 500 times in the three exercise intensities, returning to resting value during recovery. In the RRV power spectrum three components have been identified: (1) high frequency peak (HF), central frequency about 0.24 Hz at rest and recovery, and 0.28 Hz, SEM 0.02, 0.37 Hz, SEM 0.03 and 0.48 Hz, SEM 0.06 during the three exercise intensities, respectively; (2) low frequency peak (LF), central frequency about 0.1 Hz independent of the metabolic state; (3) very low frequency component (VLF), less than 0.05 Hz, no peak observed. The HF peak power, as a percentage of the total power (HF%), averaged 16%, SEM 5% at rest and did not change during exercise, whereas during recovery it decreased to 5%-10%. The LF% and VLF% were about 50% and 35% at rest and during low exercise intensity, respectively. At higher intensities, LF% decreased to 16% and VLF% increased to 70%. During recovery a return to resting values occurred. The HF component may reflect the increased respiratory rate and the LF peak changes the resetting of the baroreceptor reflex with exercise. The hypothesis is made that VLF fluctuations in heart rate might be partially mediated by the sympathetic system.