Age-related changes in regional brain mitochondria from Fischer 344 rats

Brain mitochondrial function has been posited to decline with aging. In order to test this hypothesis, cortical and striatal mitochondria were isolated from Fischer 344 rats at 2, 5, 11, 24 and 33 months of age. Mitochondrial membrane potential remained stable through 24 months, declining slightly in mitochondria from both brain regions at 33 months. The ability of calcium to induce mitochondrial swelling and depolarization, characteristics of the permeability transition, was remarkably stable through 24 months of age and increased at advanced ages only for cortical, but not striatal, mitochondria. Striatal mitochondria were more sensitive to calcium than were cortical mitochondria throughout the first 2 years of life. A two-fold increased resistance to calcium was observed in striatal mitochondria between 5 and 11 months. Although these measurements do demonstrate changes in mitochondrial function with aging, the changes in polarization are relatively small and the increased cortical susceptibility to the permeability transition only occurred at very advanced ages. Thus mitochondrial decline with advanced age depends upon brain region.     

Rapid, sequential changes in surface morphology of PC12 pheochromocytoma cells in response to nerve growth factor

The effect of nerve growth factor (NGF), a substance that promotes the differentiation and maintenance of certain neurons, was studied via scanning electron microscopy utilizing the PC12 clonal NGF-responsive pheochromocytoma cell line. After 2-4 d of exposure to NGF, these cells acquire many of the properties of normal sympathic neurons. However, by phase microscopy, no changes are discernible within the first 12-18 h. Since the primary NGF receptor appears to be a membrane receptor, it seemed likely that some of the initial responses to the factor may be surface related. PC12 cells maintained without NGF are round to ovoid and have numerous microvilli and small blebs. After the addition of NGF, there is a rapidly initiated sequential change in the cell surface. Ruffles appear over the dorsal surface of the cells with 1 min, become prominent by 3 min, and almost disappear by 7 min. Microvilli, conversely, disappear as the dorsal ruffles become prominent. Ruffles are seen at the the periphery of cell at 3 min, are prominent on most of the cells by 7 min and are gone by 15 min. The surface remains smooth from 15 min until 45 min when large blebs appear. The large blebs are present on most cells at 2 h and are gone by 4 h. The surface remains relatively smooth until 6-7 h of NGF treatment, when microvilli reappear as small knobs. These microvilli increase in both number and length to cover the cell surface by 10 h. These changes were not observed with other basic proteins, with α-bungarotoxin (which binds specifically to PC12 membranes), and were not affected by an RNA synthesis inhibitor that blocks initiation of neurite outgrowth. Changes in the cell surface architecture appear to be among the earlist NGF responses yet detected and may represent or reflect primary events in the mechanism of the factor’s action.     

The miR-17-5p microRNA is a key regulator of the G1/S phase cell cycle transition

Background

MicroRNAs are modifiers of gene expression, acting to reduce translation through either translational repression or mRNA cleavage. Recently, it has been shown that some microRNAs can act to promote or suppress cell transformation, with miR-17-92 described as the first oncogenic microRNA. The association of miR-17-92 encoded microRNAs with a surprisingly broad range of cancers not only underlines the clinical significance of this locus, but also suggests that miR-17-92 may regulate fundamental biological processes, and for these reasons miR-17-92 has been considered as a therapeutic target.

Results

In this study, we show that miR-17-92 is a cell cycle regulated locus, and ectopic expression of a single microRNA (miR-17-5p) is sufficient to drive a proliferative signal in HEK293T cells. For the first time, we reveal the mechanism behind this response - miR-17-5p acts specifically at the G1/S-phase cell cycle boundary, by targeting more than 20 genes involved in the transition between these phases. While both pro- and anti-proliferative genes are targeted by miR-17-5p, pro-proliferative mRNAs are specifically up-regulated by secondary and/or tertiary effects in HEK293T cells.

Conclusion

The miR-17-5p microRNA is able to act as both an oncogene and a tumor suppressor in different cellular contexts; our model of competing positive and negative signals can explain both of these activities. The coordinated suppression of proliferation-inhibitors allows miR-17-5p to efficiently de-couple negative regulators of the MAPK (mitogen activated protein kinase) signaling cascade, promoting growth in HEK293T cells. Additionally, we have demonstrated the utility of a systems biology approach as a unique and rapid approach to uncover microRNA function.     

Two new cytotypes reinforce that <Emphasis Type="Italic">Micronycteris hirsuta</Emphasis> Peters, 1869 does not represent a monotypic taxon

Background

The genus Micronycteris is a diverse group of phyllostomid bats currently comprising 11 species, with diploid number (2n) ranging from 26 to 40 chromosomes. The karyotypic relationships within Micronycteris and between Micronycteris and other phyllostomids remain poorly understood. The karyotype of Micronycteris hirsuta is of particular interest: three different diploid numbers were reported for this species in South and Central Americas with 2n?=?26, 28 and 30 chromosomes. Although current evidence suggests some geographic differentiation among populations of M. hirsuta based on chromosomal, morphological, and nuclear and mitochondrial DNA markers, the recognition of new species or subspecies has been avoided due to the need for additional data, mainly chromosomal data.

Results

We describe two new cytotypes for Micronycteris hirsuta (MHI) (2n?=?26 and 25, NF?=?32), whose differences in diploid number are interpreted as the products of Robertsonian rearrangements. C-banding revealed a small amount of constitutive heterochromatin at the centromere and the NOR was located in the interstitial portion of the short arm of a second pair, confirmed by FISH. Telomeric probes hybridized to the centromeric regions and weakly to telomeric regions of most chromosomes. The G-banding analysis and chromosome painting with whole chromosome probes from Carollia brevicauda (CBR) and Phyllostomus hastatus (PHA) enabled the establishment of genome-wide homologies between MHI, CBR and PHA.

Conclusions

The karyotypes of Brazilian specimens of Micronycteris hirsuta described here are new to Micronycteris and reinforce that M. hirsuta does not represent a monotypic taxon. Our results corroborate the hypothesis of karyotypic megaevolution within Micronycteris, and strong evidence for this is that the entire chromosome complement of M. hirsuta was shown to be derivative with respect to species compared in this study.

     

Differential expression of specific sulphate transporters underlies seasonal and spatial patterns of sulphate allocation in trees

Sulphate uptake and its distribution within plants depend on the activity of different sulphate transporters (SULTR). In long‐living deciduous plants such as trees, seasonal changes of spatial patterns add another layer of complexity to the question of how the interplay of different transporters adjusts S distribution within the plant to environmental changes. Poplar is an excellent model to address this question because its S metabolism is already well characterized. In the present study, the importance of SULTRs for seasonal sulphate storage and mobilization was examined in the wood of poplar (Populus tremula × P. alba) by analysing their gene expression in relation to sulphate contents in wood and xylem sap. According to these results, possible functions of the respective SULTRs for seasonal sulphate storage and mobilization in the wood are suggested. Together, the present results complement the previously published model for seasonal sulphate circulation between leaves and bark and provide information for future mechanistic modelling of whole tree sulphate fluxes.     

Structural and functional differences between porcine brain and budding yeast microtubules

The cytoskeleton of eukaryotic cells relies on microtubules to perform many essential functions. We have previously shown that, in spite of the overall conservation in sequence and structure of tubulin subunits across species, there are differences between mammalian and budding yeast microtubules with likely functional consequences for the cell. Here we expand our structural and function comparison of yeast and porcine microtubules to show different distribution of protofilament number in microtubules assembled in vitro from these two species. The different geometry at lateral contacts between protofilaments is likely due to a more polar interface in yeast. We also find that yeast tubulin forms longer and less curved oligomers in solution, suggesting stronger tubulin:tubulin interactions along the protofilament. Finally, we observed species-specific plus-end tracking activity for EB proteins: yeast Bim1 tracked yeast but not mammalian MTs, and human EB1 tracked mammalian but not yeast MTs. These findings further demonstrate that subtle sequence differences in tubulin sequence can have significant structural and functional consequences in microtubule structure and behavior.     

Changes in plasma volume during bed rest: effects of menstrual cycle and estrogen administration

Bed rest (BR) is associated with a decrease in plasma volume (PV), which may contribute to the impaired orthostatic and exercise tolerances seen immediately after BR. The purpose of this study was to determine whether increases in blood estrogen concentration, either during normal menstrual cycles or during exogenous estrogen administration, would attenuate this loss of PV. Nineteen healthy women (21-39 yr of age) completed the study. Twelve women underwent duplicate 11-day BR without estrogen supplementation. PV decreased significantly (P less than or equal to 0.01) during both BR's, from 2,531 +/- 113 to 2,027 +/- 102 ml during BR1 and from 2,445 +/- 115 to 2,244 +/- 96 ml during BR2. The women who began BR in the periovulatory stage of the menstrual cycle (n = 3), a time of elevated endogenous estrogens, had a transient delay in loss of PV during the first 5 days of BR. Women who began BR during other stages of the menstrual cycle (n = 17) showed the established trend to decrease PV primarily during the first few days of BR. Seven additional women underwent a single 12-day BR while taking estrogen supplementation (1.25 mg/day premarin). PV decreased during the first 4-5 days of BR, then returned toward the pre-BR level during the remainder of the BR (pre-BR PV, 2,525 +/- 149 ml; post-BR PV, 2,519 +/- 162 ml). Thus menstrual fluctuations in endogenous estrogens appear to have only small transient effects on the loss of PV during BR, whereas exogenous estrogen supplementation significantly attenuates PV loss.     

Effect of exercise hemoconcentration and hyperosmolality on exercise responses

We investigated the effects of a decrease in plasma volume (PV) and an increase in plasma osmolality during exercise on circulatory and thermoregulatory responses. Six subjects cycled at approximately 65% of their maximum O2 uptake in a warm environment (30 degrees C, 40% relative humidity). After 30 min of control (C) exercise (no infusion), PV decreased 13.0%, or 419 +/- 106 (SD) ml, heart rate (HR) increased to 167 +/- 3 beats/min, and esophageal temperature (Tes) rose to 38.19 +/- 0.09 degrees C (SE). During infusion studies (INF), infusates were started after 10 min of exercise. The infusates contained 5% albumin suspended in 0.45, 0.9, or 3.0% saline. The volume of each infusate was adjusted so that during the last 10 min of exercise PV was maintained at the preexercise level and osmolality was allowed to differ. HR was significantly lower (10-16 beats/min) during INF than during C. Tes was reduced significantly during INF, with trends for increased skin blood flow and decreased sweating rates. No significant differences in HR, Tes, or sweating rate occurred between the three infusion conditions. We conclude that the decrease in PV, which normally accompanies moderate cycle exercise, compromises circulatory and thermal regulations. Increases in osmolality appear to have small if any effects during such short-term exercise.