Changes in fiber composition of soleus muscle during rat hindlimb suspension

Chronic reduction of gravitational load in the rear limbs of rats to simulate the influence of near-zero gravity in skeletal muscles has been shown previously to elicit atrophy in the soleus muscle. Use of this model by the present investigation indicates that soleus atrophy was characterized by a decline in the number of fibers in groups that contained the slow isoenzyme of myosin and which were classified as type I from intensity of staining to myofibrillar actomyosin adenosinetriphosphatase (ATPase) and to NADH tetrazolium reductase. Furthermore total fiber number was not changed, whereas fibers containing the intermediate isoenzyme and those classified as type IIa increased. There results could be explained by either a change in the composition within existing fibers or a simultaneous loss of slow fibers and de novo synthesis of intermediate and fast fibers. Evidence for transformation included an absence of embryonic or neonatal myosin in muscles from suspended rats and the constant fiber number that was unchanged by 4 wk of suspension. Furthermore although fiber areas of both groups of type I and IIa fibers declined during suspension, variability of the fiber areas within each group did not increase.     

Quantifying Biomass Changes of Single CD8+ T Cells during Antigen Specific Cytotoxicity

Existing approaches that quantify cytotoxic T cell responses rely on bulk or surrogate measurements which impede the direct identification of single activated T cells of interest. Single cell microscopy or flow cytometry methodologies typically rely on fluorescent labeling, which limits applicability to primary cells such as human derived T lymphocytes. Here, we introduce a quantitative method to track single T lymphocyte mediated cytotoxic events within a mixed population of cells using live cell interferometry (LCI), a label-free microscopy technique that maintains cell viability. LCI quantifies the mass distribution within individual cells by measuring the phase shift caused by the interaction of light with intracellular biomass. Using LCI, we imaged cytotoxic T cells killing cognate target cells. In addition to a characteristic target cell mass decrease of 20–60% over 1–4 h following attack by a T cell, there was a significant 4-fold increase in T cell mass accumulation rate at the start of the cytotoxic event and a 2–3 fold increase in T cell mass relative to the mass of unresponsive T cells. Direct, label-free measurement of CD8+ T and target cell mass changes provides a kinetic, quantitative assessment of T cell activation and a relatively rapid approach to identify specific, activated patient-derived T cells for applications in cancer immunotherapy.     

Isomeric lipid signatures reveal compartmentalized fatty acid metabolism in cancer

The cellular energy and biomass demands of cancer drive a complex dynamic between uptake of extracellular FAs and their de novo synthesis. Given that oxidation of de novo synthesized FAs for energy would result in net-energy loss, there is an implication that FAs from these two sources must have distinct metabolic fates; however, hitherto, all FAs have been considered part of a common pool. To probe potential metabolic partitioning of cellular FAs, cancer cells were supplemented with stable isotope-labeled FAs. Structural analysis of the resulting glycerophospholipids revealed that labeled FAs from uptake were largely incorporated to canonical (sn-) positions on the glycerol backbone. Surprisingly, labeled FA uptake also disrupted canonical isomer patterns of the unlabeled lipidome and induced repartitioning of n-3 and n-6 PUFAs into glycerophospholipid classes. These structural changes support the existence of differences in the metabolic fates of FAs derived from uptake or de novo sources and demonstrate unique signaling and remodeling behaviors usually hidden from conventional lipidomics.     

Identification and functional analysis of NOL7 nuclear and nucleolar localization signals

Background  

NOL7 is a candidate tumor suppressor that localizes to a chromosomal region 6p23. This locus is frequently lost in a number of malignancies, and consistent loss of NOL7 through loss of heterozygosity and decreased mRNA and protein expression has been observed in tumors and cell lines. Reintroduction of NOL7 into cells resulted in significant suppression of in vivo tumor growth and modulation of the angiogenic phenotype. Further, NOL7 was observed to localize to the nucleus and nucleolus of cells. However, the mechanisms regulating its subcellular localization have not been elucidated.     

Solution Radioactivated by Hadron Radiation Can Increase Sister Chromatid Exchanges

When energetic particles irradiate matter, it becomes activated by nuclear reactions. Radioactivation induced cellular effects are not clearly understood, but it could be a part of bystander effects. This investigation is aimed at understanding the biological effects from radioactivation in solution induced by hadron radiation. Water or phosphate buffered saline was activated by being exposed to hadron radiation including protons, carbon- and iron-ions. 1 mL of radioactivated solution was transferred to flasks with Chinese hamster ovary (CHO) cells cultured in 5 mL of complete media. The induction of sister chromatid exchanges (SCE) was used to observe any increase in DNA damage responses. The energy spectrum and the half-lives of the radioactivation were analyzed by NaI scintillation detector in order to identify generated radionuclides. In the radioactivated solution, 511 keV gamma-rays were observed, and their half-lives were approximately 2 min, 10 min, and 20 min. They respectively correspond to the beta+ decay of ¹⁵O, ¹³N, and ¹¹C. The SCE frequencies in CHO cells increased depending on the amount of radioactivation in the solution. These were suppressed with a 2-hour delayed solution transfer or pretreatment with dimethyl sulfoxide (DMSO). Our results suggest that the SCE induction by radioactivated solution was mediated by free radicals produced by the annihilated gamma-rays. Since the SCE induction and DMSO modulation are also reported in radiation-induced bystander effects, our results imply that radioactivation of the solution may have some contribution to the bystander effects from hadron radiation. Further investigations are required to assess if radioactivation effects would attribute an additional level of cancer risk of the hadron radiation therapy itself.     

Natal recruitment and adult retention in a population of nine-banded armadillos

Nine-banded armadillosDasypus novemcinctus Linnaeus, 1758 are interesting in part because (a) they give birth to litters of genetically identical quadruplets, and (b) the species’ range has expanded rapidly throughout the southern United States during the last 150 years, suggesting substantial dispersal of individuals. Using data from 7 field seasons between 1992 and 1999, we examined the extent of juvenile recruitment and retention of adults in a population of armadillos from northern Florida. There were no sex differences in the likelihood of recruitment or most attributes of male and female recruits at any age. In the few cases where more than one littermate was recruited into the population, siblings were significantly more widely dispersed as adults than they were as juveniles, thus limiting opportunities for interaction among clonal siblings. There was some evidence that recruits ranged more widely than other individuals, suggesting recruits may have been searching for suitable sites to establish themselves. Recruits were heavier than non-recruits as both juveniles and yearlings, which may have aided in establishing a home range, but recruits were lighter than other animals as adults. Overall, slightly less than 50% of armadillos first captured as adults were never seen in a subsequent year, suggesting these individuals may have been transients. However, some adults remained in the population for multiple years, moving very little from the area where they were first sighted. As with recruits, there were no sex differences in the likelihood of adults being retained in the population nor in the attributes of retained males and females. Retained animals exhibited more extensive anatomical damage and moved farther between successive sightings within years than did non-residents. Adults were more likely to be retained in the population than juveniles were to be recruited, and retained adults were older, heavier, and exhibited more extensive anatomical damage than did recruits. Our data seem to indicate a population characterized by limited recruitment of juveniles (particularly of clonemates) and an adult population exhibiting considerable turnover from year to year, but with a core of individuals who are long-term residents.     

Homeostatic control of presynaptic release is triggered by postsynaptic membrane depolarization.

Homeostatic mechanisms regulate synaptic function to maintain nerve and muscle excitation within reasonable physiological limits. The mechanisms that initiate homeostasic changes to synaptic function are not known. We specifically impaired cellular depolarization by expressing the Kir2.1 potassium channel in Drosophila muscle. In Kir2.1-expressing muscle there is a persistent outward potassium current ( approximately 10 nA), decreased muscle input resistance (50-fold), and a hyperpolarized resting potential. Despite impaired muscle excitability, synaptic depolarization of muscle achieves wild-type levels. A quantal analysis demonstrates that increased presynaptic release (quantal content), without a change in quantal size (mEPSC amplitude), compensates for altered muscle excitation. Because morphological synaptic growth is normal, we conclude that a homeostatic increase in presynaptic release compensates for impaired muscle excitability. These data demonstrate that a monitor of muscle membrane depolarization is sufficient to initiate synaptic homeostatic compensation.     

Blueberry flavonoids inhibit matrix metalloproteinase activity in DU145 human prostate cancer cells.

Regulation of the matrix metalloproteinases (MMPs), the major mediators of extracellular matrix (ECM) degradation, is crucial to regulate ECM proteolysis, which is important in metastasis. This study examined the effects of 3 flavonoid-enriched fractions (a crude fraction, an anthocyanin-enriched fraction, and a proanthocyanidin-enriched fraction), which were prepared from lowbush blueberries (Vaccinium angustifolium), on MMP activity in DU145 human prostate cancer cells in vitro. Using gelatin gel electrophoresis, MMP activity was evaluated from cells after 24-hr exposure to blueberry fractions. All fractions elicited an ability to decrease the activity of MMP-2 and MMP-9. Of the fractions tested, the proanthocyanidin-enriched fraction was found to be the most effective at inhibiting MMP activity in these cells. No induction of either necrotic or apoptotic cell death was noted in these cells in response to treatment with the blueberry fractions. These findings indicate that flavonoids from blueberry possess the ability to effectively decrease MMP activity, which may decrease overall ECM degradation. This ability may be important in controlling tumor metastasis formation.     

Evidence for expression of a common myosin heavy chain phenotype in future fast and slow skeletal muscle during initial stages of avian embryogenesis

We have utilized a key biochemical determinant of muscle fiber type, myosin isoform expression, to investigate the initial developmental program of future fast and slow skeletal muscle fibers. We examined myosin heavy chain (HC) phenotype from the onset of myogenesis in the limb bud muscle masses of the chick embryo through the differentiation of individual fast and slow muscle masses, as well as in newly formed myotubes generated in adult muscle by weight overload. Myosin HC isoform expression was analyzed by immunofluorescence localization with a battery of anti-myosin antibodies and by electrophoretic separation with SDS-PAGE. Results showed that the initial myosin phenotype in all skeletal muscle cells formed during the embryonic period (until at least 8 days in ovo) consisted of expression of a myosin HC which shares antigenic and electrophoretic migratory properties with ventricular myosin and a distinct myosin HC which shares antigenic and electrophoretic migratory properties with fast skeletal isomyosin. Similar results were observed in newly formed myotubes in adult muscle. Future fast and slow muscle fibers could only be discriminated from each other in developing limb bud muscles by the onset of expression of slow skeletal myosin HC at 6 days in ovo. Slow skeletal myosin HC was expressed only in myotubes which became slow fibers. These findings suggest that the initial commitment of skeletal muscle progenitor cells is to a common skeletal muscle lineage and that commitment to a fiber-specific lineage may not occur until after localization of myogenic cells in appropriate premuscle masses. Thus, the process of localization, or events which occur soon thereafter, may be involved in determining fiber type.     

Extrachromosomal Elements Cause a Reduced Division Potential in Nib 1 Strains of Saccharomyces Cerevisiae

The nib 1 allele of yeast confers a sensitivity to an endogenous plasmid, 2 mu DNA, in that nib 1 strains bearing 2 mu DNA (cir+) exhibit a reduction in division potential. In the present study, the reduction in division potential characteristic of nib 1 cir+ strains is shown to be dependent on the simultaneous presence of both the A and the D open reading frames of 2 mu DNA as well as on the presence of an unidentified extrachromosomal element other than 2 mu DNA. Furthermore, in nib 1 strains, an uncharacterized extrachromosomal element can cause a less severe reduction of division potential in the absence of intact 2 mu DNA. Thus, the nib 1 allele may confer a generalized sensitivity to extrachromosomal elements.