Proliferative capacity and DNA content of aging human diploid cells in culture: A cytophotometric and autoradiographic analysis

Using a combination of DNA-cytophotometry and tritiated thymidine-autoradiography, we have shown that the majority of nondividing cells in serially propagated human diploid cell populations have the 2C DNA content consistent with their being arrested in the G₁ phase of the diploid cell cycle. Unlabeled 4C cells appear increasingly with time in culture. These may be arrested G₂ diploids or they may be G₁ tetraploids, since there is an associated increase in polyploidy in older cultures as evidenced by the appearance of labeled 8C cells.     

Pavlovian conditioning: pigeon nictitating membrane

A new Pavlovian conditioning preparation was developed using the nictitating membrane of the restrained pigeon. Either visual or auditory stimuli served as conditioned stimuli (CSs) with an unconditioned stimulus (US) of a puff of air to the cornea. Movement of the nictitating membrane constituted the conditioned and unconditioned responses (CR and UR). Conditioning was studied with the Kamin blocking procedure. In agreement with findings from other conditioning preparations, responding to the redundant stimulus was attenuated relative to a stimulus that received the same number of CS-US pairings in a compound-conditioning procedure. Although response attenuation occurred, substantial individual variation was observed within the blocking procedure, a finding with some precedent in the experimental literature. Theoretical analysis and neural-network simulations indicate that inter-subject variation in response attenuation may result from differences in the extent to which contextual stimuli contribute to the functional CS.     

A protein with an inactive pterin‐4a‐carbinolamine dehydratase domain is required for Rubisco biogenesis in plants

Ribulose‐1,5‐bisphosphate carboxylase/oxygenase (Rubisco) plays a critical role in sustaining life by catalysis of carbon fixation in the Calvin–Benson pathway. Incomplete knowledge of the assembly pathway of chloroplast Rubisco has hampered efforts to fully delineate the enzyme's properties, or seek improved catalytic characteristics via directed evolution. Here we report that a Mu transposon insertion in the Zea mays (maize) gene encoding a chloroplast dimerization co‐factor of hepatocyte nuclear factor 1 (DCoH)/pterin‐4α‐carbinolamine dehydratases (PCD)‐like protein is the causative mutation in a seedling‐lethal, Rubisco‐deficient mutant named Rubisco accumulation factor 2 (raf2‐1). In raf2 mutants newly synthesized Rubisco large subunit accumulates in a high‐molecular weight complex, the formation of which requires a specific chaperonin 60‐kDa isoform. Analogous observations had been made previously with maize mutants lacking the Rubisco biogenesis proteins RAF1 and BSD2. Chemical cross‐linking of maize leaves followed by immunoprecipitation with antibodies to RAF2, RAF1 or BSD2 demonstrated co‐immunoprecipitation of each with Rubisco small subunit, and to a lesser extent, co‐immunoprecipitation with Rubisco large subunit. We propose that RAF2, RAF1 and BSD2 form transient complexes with the Rubisco small subunit, which in turn assembles with the large subunit as it is released from chaperonins.     

Inflammatory expression profiles in monocyte-to-macrophage differentiation in patients with systemic lupus erythematosus and relationship with atherosclerosis

Introduction

Our objectives were to examine mononuclear cell gene expression profiles in patients with systemic lupus erythematosus (SLE) and healthy controls and to compare subsets with and without atherosclerosis to determine which genes’ expression is related to atherosclerosis in SLE.

Methods

Monocytes were obtained from 20 patients with SLE and 16 healthy controls and were in vitro-differentiated into macrophages. Subjects also underwent laboratory and imaging studies to evaluate for subclinical atherosclerosis. Whole-genome RNA expression microarray was performed, and gene expression was examined.

Results

Gene expression profiling was used to identify gene signatures that differentiated patients from controls and individuals with and without atherosclerosis. In monocytes, 9 out of 20 patients with SLE had an interferon-inducible signature compared with 2 out of 16 controls. By looking at gene expression during monocyte-to-macrophage differentiation, we identified pathways which were differentially regulated between SLE and controls and identified signatures based on relevant intracellular signaling molecules which could differentiate SLE patients with atherosclerosis from controls. Among patients with SLE, we used a previously defined 344-gene atherosclerosis signature in monocyte-to-macrophage differentiation to identify patient subgroups with and without atherosclerosis. Interestingly, this signature further classified patients on the basis of the presence of SLE disease activity and cardiovascular risk factors.

Conclusions

Many genes were differentially regulated during monocyte-to-macrophage differentiation in SLE patients compared with controls. The expression of these genes in mononuclear cells is important in the pathogenesis of SLE, and molecular profiling using gene expression can help stratify SLE patients who may be at risk for development of atherosclerosis.     

Isolating the role of elevated Phlda2 in asymmetric late fetal growth restriction in mice

Pleckstrin homology-like domain family A member 2 (PHLDA2) is a maternally expressed imprinted gene whose elevated expression has been linked to fetal growth restriction in a number of human studies. In mice, Phlda2 negatively regulates placental growth and limits the accumulation of placental glycogen. We previously reported that a three-copy transgene spanning the Phlda2 locus drove a fetal growth restriction phenotype late in gestation, suggesting a causative role for PHLDA2 in human growth restriction. However, in this mouse model, Phlda2 was overexpressed by fourfold, alongside overexpression of a second imprinted gene, Slc22a18. Here, we genetically isolate the role of Phlda2 in driving late fetal growth restriction in mice. We furthermore show that this Phlda2-driven growth restriction is asymmetrical, with a relative sparing of the brain, followed by rapid catch-up growth after birth, classic features of placental insufficiency. Strikingly, fetal growth restriction showed strain-specific differences, being apparent on the 129S2/SvHsd (129) genetic background and absent on the C57BL6 (BL6) background. A key difference between these two strains is the placenta. Specifically, BL6 placentae possess a more extensive endocrine compartment and substantially greater stores of placental glycogen. Taken together, these data support a direct role for elevated Phlda2 in limiting fetal growth but also suggest that growth restriction only manifests when there is limited placental reserve. These findings should be taken into account in interpreting the results from human studies.KEY WORDS: Phlda2, Fetal growth restriction, Asymmetric     

Assessing the Bioavailability and Risk from Metal-Contaminated Soils and Dusts

Exposure to contaminated soil and dust is an important pathway in human health risk assessment. Physical and chemical characteristics and biological factors determine the bioaccessibility/bioavailability of soil and dust contaminants. Within a single sample, contamination may arise from multiple sources of toxic elements that may exist as different species that impact bioavailability. In turn, the bioaccessibility/bioavailability of soil and dust contaminants directly impacts human health risk. Research efforts focusing on development and application of in vitro and in vivo methods to measure the bioaccessibility/bioavailability of metal-contaminated soils have advanced in recent years. The objective of this workshop was to focus on developments in assessing the bioaccessibility/bioavailability of arsenic-contaminated soils, metals’ contamination in urban Canadian residences and potential children's exposures to toxic elements in house dust, an urban community-based study (i.e., West Oakland Residential Lead Assessment), bioavailability studies of soil cadmium, chromium, nickel, and mercury and human exposures to contaminated Brownfield soils. These presentations covered issues related to human health and bioavailability along with the most recent studies on community participation in assessing metals’ contamination, studies of exposures to residential contamination, and in vitro and in vivo methods development for assessing the bioaccessibility/bioavailability of metals in soils and dusts.     

Association between S100B Levels and Long-Term Outcome after Aneurysmal Subarachnoid Hemorrhage: Systematic Review and Pooled Analysis

S100 calcium binding protein B (S100B), a well-studied marker for neurologic injury, has been suggested as a candidate for predicting outcome after subarachnoid hemorrhage. We performed a pooled analysis summarizing the associations between S100B protein in serum and cerebrospinal fluid (CSF) with radiographic vasospasm, delayed ischemic neurologic deficit (DIND), delayed cerebral infarction, and Glasgow Outcome Scale (GOS) outcome. A literature search using PubMed, the Cochrane Library, and the EMBASE databases was performed to identify relevant studies published up to May 2015. The weighted Stouffer’s Z method was used to perform a pooled analysis of outcome measures with greater than three studies. A total of 13 studies were included in this review. Higher serum S100B level was found to be associated with cerebral infarction as diagnosed by CT (p_adj = 3.1 x 10⁻⁴) and worse GOS outcome (p_adj = 5.5 x 10⁻¹¹). There was no association found between serum and CSF S100B with radiographic vasospasm or DIND. S100B is a potential prognostic marker for aSAH outcome.