Complementation in heterokaryons derived from a thymidine kinase deficient cell line and senescent human diploid cells.

Incorporation of [3H]thymidine was observed in both parental nuclei in heterokaryons resulting from the fusion of post-mitotic, "senescent" human diploid cells and a thymidine kinase-deficient murine cell line (3T3der-4E). The senescent nuclei displayed a sudden increase of activity approximately 4--6 hours after fusion. A moderate increase of thymidine incorporation was observed in 3T3der-4E cells cocultivated with but not fused to senescent cells, consistent with metabolic cooperation. Chromosome preparations of cultures fixed approximately 24 hr after fusion revealed the presence of hybrid metaphase cells containing almost the entire human complement. All of the identifiable human chromosomes were bi-armed, suggesting that the senescent nuclei were stimulated to reinitiate replicative DNA synthesis rather than repair synthesis in these heterokaryons.     

Assembly and Solution Structure of the Core Retromer Protein Complex

Retromer is a peripheral membrane protein complex that has pleiotropic roles in endosomal membrane trafficking. The core of retromer possesses three subunits, VPS35, VPS29 and VPS26, that play different roles in binding to cargo, regulatory proteins and complex stabilization. We have performed an investigation of the thermodynamics of core retromer assembly using isothermal titration calorimetry (ITC) demonstrating that VPS35 acts as the central subunit to which VPS29 and VPS26 bind independently. Furthermore, we confirm that the conserved PRLYL motif of the large VPS35 subunit is critical for direct VPS26 interaction. Heat capacity measurements of VPS29 and VPS26 binding to VPS35 indicate extensive binding interfaces and suggest conformational alterations in VPS29 or VPS35 upon complex formation. Solution studies of the retromer core using small‐angle X‐ray scattering allow us to propose a model whereby VPS35 forms an extended platform with VPS29 and VPS26 bound at distal ends, with the potential for forming dimeric assemblies.     

The relationship between the rate of entry into S phase, concentration of DNA polymerase alpha, and cell volume in human diploid fibroblast-like monokaryon cells

We have examined the kinetic relationship between the rate of entry into the S phase in human diploid fibroblast-like (HDFL) monokaryon cells and (1) the concentration of DNA polymerase alpha activity and (2) the cell volume. In the former studies, a first-order dependence between the rate of entry into the S phase and the concentration of DNA polymerase alpha activity was observed, consistent with the enzyme, or a coregulated factor, being rate limiting for this metabolic process. Examination of the nature of the dependence of the rate of entry into the S phase upon cell volume revealed a more complex relationship. The results obtained in studies with synchronized cultures are consistent with the presence of two to three rate-limiting reactants when cell volume is the independent variable. Studies with asynchronous HDFL cell cultures revealed that the smallest cells in the G1 population, presumably the early G1 cells, enter the S phase at an increasing rate as a function of cell volume up to a certain size, beyond which the cells enter at a decreasing rate similar to that observed in the studies with the synchronized cultures. Similar studies examining the relationship between cell volume and the rate of entry into S phase in three established immortal cell lines revealed positive correlation between the rate of entry into S phase and cell volume throughout the size range of the G1 population. This latter observation suggests that the factors involved in the initiation of the S phase may be present in concentrations that are not rate limiting in immortal cell lines.     

Murine temperature-sensitive DNA polymerase α mutant displays a diminished capacity to stimulate DNA synthesis in senescent human fibroblast nuclei in heterokaryons at the nonpermissive condition

We have investigated the capacity of a murine cell line with a temperature-sensitive (ts) mutation in the DNA polymerase α (Pola) locus and a series of ts non-Pola mutant cell lines from separate complementation groups to stimulate DNA synthesis, in senescent fibroblast nuclei in heterokaryons. In the Pola mutant × senescent heterodikaryons, both human and murine nuclei display significantly diminished levels of DNA synthesis at the restrictive temperature (39.5°C) as determined by [³H]thymidine labeling in autoradiographs. In contrast, all of the non-Pola mutants, as well as the parental (wild-type) murine cells, induced similar levels of DNA synthesis in both parental nuclei at the nonpermissive and permissive temperatures. Similarly, young human fibroblasts are also able to initiate DNA synthesis in heterokaryons with the ts Pola mutant at the two temperatures. In order to determine if complementation of the non-Pola mutants requires induction of serum responsive factors in the senescent cells, fusion studies of similar design were conducted with young and old human fibroblasts incubated in low serum (0.2%) for 48 hr prior to and after cell fusion. Again, a diminished level of DNA synthesis was observed at 39.5°C in the Pola mutant x senescent cell heterokaryons. In these low-serum studies, both parental nuclei in the Pola x young cell heterokaryons and the human nuclei in heterokaryons with one of the non-Pola mutants (FT107) also displayed diminished levels of DNA synthetic activity. All of the other mutants are able to support similar levels of synthetic activity at both temperatures in the presence of reduced serum. The nature of the mutation in three of the non-Pola lines has not been determined but, like the Pola mutant cells, are inhibited in the G₁ phase of the cell cycle when incubated at the nonpermissive temperature (39.5°C). The fourth non-Pola mutant line is known to have at least one ts mutation in the cdc2 gene and is inhibited in the G₂ phase when exposed to 39.5°C. These results suggest that there may be a functional deficiency of pol α in senescent human fibroblasts, and this replication factor may be one of the rate-limiting factors involved in loss of the capacity to initiate DNA synthesis in senescent cells. © 1994 Wiley-Liss, Inc.     

N-methyl D-aspartate receptor-mediated bidirectional control of extracellular signal-regulated kinase activity in cortical neuronal cultures

N-Methyl D-aspartate (NMDA) receptor activation of extracellular-signal regulated kinase (ERK) was examined in primary cortical cultures. Tetrodotoxin, NMDA receptor antagonists, or reduced extracellular calcium (0.1 mm) greatly decreased basal levels of phospho-ERK2, indicating that activity-dependent activation of NMDA receptors maintained a high level of basal ERK2 activation. This activity-dependent activation of phospho-ERK2 was blocked by pertussis toxin and inhibition of calcium/calmodulin-dependent kinase II and phosphatidylinositol 3-kinase but not by inhibition of protein kinase C or cAMP-dependent protein kinase. Addition of a calcium ionophore or 100 microm NMDA decreased phospho-ERK2 in the presence of 1 mm extracellular calcium but enhanced phospho-ERK2 in 0.1 mm extracellular calcium. The reduction in basal phospho-ERK2 by 100 microm NMDA was also reflected as a decrease in phospho-cAMP response element-binding protein. Inhibition of tyrosine phosphatases and serine/threonine phosphatases protein phosphatase 1 (PP1), PP2A, and PP2B did not prevent the inhibitory effect of NMDA. In the presence of tetrodotoxin, NMDA produced a bell-shaped dose-response curve with stimulation of phospho-ERK2 at 10, 25, and 50 microm NMDA and reduced stimulation at 100 microm NMDA. NMDA (50 microm) stimulation of phospho-ERK2 was completely blocked by pertussis toxin and inhibitors of phosphatidylinositol 3-kinase and was partially blocked by a calcium/calmodulin-dependent kinase II inhibitor. These results suggests that NMDA receptors can bidirectionally control ERK signaling.     

Transgene expression patterns indicate that spaceflight affects stress signal perception and transduction in arabidopsis

The use of plants as integral components of life support systems remains a cornerstone of strategies for long-term human habitation of space and extraterrestrial colonization. Spaceflight experiments over the past few decades have refined the hardware required to grow plants in low-earth orbit and have illuminated fundamental issues regarding spaceflight effects on plant growth and development. Potential incipient hypoxia, resulting from the lack of convection-driven gas movement, has emerged as a possible major impact of microgravity. We developed transgenic Arabidopsis containing the alcohol dehydrogenase (Adh) gene promoter linked to the beta-glucuronidase (GUS) reporter gene to address specifically the possibility that spaceflight induces the plant hypoxia response and to assess whether any spaceflight response was similar to control terrestrial hypoxia-induced gene expression patterns. The staining patterns resulting from a 5-d mission on the orbiter Columbia during mission STS-93 indicate that the Adh/GUS reporter gene was activated in roots during the flight. However, the patterns of expression were not identical to terrestrial control inductions. Moreover, although terrestrial hypoxia induces Adh/GUS expression in the shoot apex, no apex staining was observed in the spaceflight plants. This indicates that either the normal hypoxia response signaling is impaired in spaceflight or that spaceflight inappropriately induces Adh/GUS activity for reasons other than hypoxia.     

Evaluation of the Cepheid GeneXpert system for detecting Bacillus anthracis

AIMS: The Cepheid GeneXpert is a four-site, automated sample preparation and real-time PCR detection system. In this study, the capability of the GeneXpert to isolate and detect nucleic acid from Bacillus anthracis Ames spores was assessed. METHODS AND RESULTS: A four-plex, dried-down bead cartridge containing PCR reagents specific for the pXO1 and pXO2 plasmids as well as sample processing and inhibition controls was evaluated. For B. anthracis Ames spores harbouring pXO1 and pXO2, samples containing 68 CFU per ml (148 spores per ml) were positive in all four replicates. A limited cross-reactivity panel, which included closely related Bacillus species, was also tested to determine the specificity of the pXO1 and pXO2 assays. No cross-reactivity occurred. Further, B. anthracis Sterne spore samples were analysed to compare results when processed using the GeneXpert to those run directly on the Cepheid SmartCycler without sample processing. The GeneXpert detection capability was three logs lower than the SmartCycler indicating the benefit of incorporating a nucleic acid extraction procedure. CONCLUSIONS: This study demonstrates that the GeneXpert is a rapid and reliable system for simultaneously detecting the B. anthracis virulence plasmids pXO1 and pXO2. SIGNIFICANCE AND IMPACT OF THE STUDY: The GeneXpert is the only platform currently available that is capable of both nucleic acid purification and real-time PCR detection enclosed within a single system. Further, all sample manipulations are automated, thus reducing errors associated with manual processing.     

Effects of Metal Mixtures on Aquatic Biota: A Review of Observations and Methods

A brief review of the historical development of metal mixture interaction analyses is presented. The two major classifications of mixture models are outlined, the “Concentration Addition” and the “Response Addition” approaches. Within these two categories, a number of graphical, mathematical and statistical methods have been used, such as the toxic unit approach, relative potencies, toxicity equivalence factors, and dose-response relationships that have been described using several methods such as probit, logit, and regression analyses. A database was generated to evaluate the frequency of occurrence of less than additive, strictly additive, and more than additive responses to metal mixture effects reported in the literature. The three responses occurred at 43, 27, and 29%, respectively. The database is available electronically from the lead author. The research required to determine the most appropriate methods to quantify the effects of metal mixtures in an ecological risk assessment (ERA) framework is discussed. Until this research is completed, ERAs should use existing models such as the toxic unit or the effects addition approach. Bioaccumulation measurements by organisms for which the accumulation to response relationship is known would also be a useful complement.     

Histone Deacetylase 3 Is Necessary for Proper Brain Development

The functional role of histone deacetylase 3 (HDAC3) in the developing brain has yet to be elucidated. We show that mice lacking HDAC3 in neurons and glia of the central nervous system, Nes-Cre/HDAC3 conditional KO mice, show major abnormalities in the cytoarchitecture of the neocortex and cerebellum and die within 24 h of birth. Later-born neurons do not localize properly in the cortex. A similar mislocalization is observed with cerebellar Purkinje neurons. Although the proportion of astrocytes is higher than normal, the numbers of oligodendrocytes are reduced. In contrast, conditional knockout of HDAC3 in neurons of the forebrain and certain other brain regions, using Thy1-Cre and calcium/calmodulin dependent protein kinase II α-Cre for ablation, produces no overt abnormalities in the organization of cells within the cortex or of cerebellar Purkinje neurons at birth. However, both lines of conditional knockout mice suffer from progressive hind limb paralysis and ataxia and die around 6 weeks after birth. The mice display an increase in overall numbers of cells, higher numbers of astrocytes, and Purkinje neuron degeneration. Taken together, our results demonstrate that HDAC3 plays an essential role in regulating brain development, with effects on both neurons and glia in different brain regions.