Specific ATM-mediated phosphorylation dependent on radiation quality

Whalen, M. K., Gurai, S. K., Zahed-Kargaran, H. and Pluth, J. M. Specific ATM-Mediated Phosphorylation Dependent on Radiation Quality. Radiat. Res. 170, 353-364 (2008).To determine whether the physical differences between high- and low-LET radiation are reflected in the biological responses of exposed cells, we detailed phospho-protein profiles of three proteins functional in radiation repair and signal transduction. Detailing gamma-H2AX, pATF2 Ser(490/498) and pSMC1 Ser(957) kinetics after X-ray and iron-ion exposure also provides a window into understanding the underlying cellular responses. Phosphorylated forms of these proteins have been documented to co-localize at sites of double-strand breaks (DSBs) after low-LET radiation exposures, and two of these phosphorylations, pATF2 and pSMC1, are specifically dependent on ATM. Flow cytometry-based methods were used to quantify total levels of each phospho-protein at various times after irradiation. As expected, we observed a greater induction and persistence in gamma-H2AX after iron-ion (high-LET) exposure compared to X-ray (low-LET) exposure. In contrast, pATF2 and pSMC1 showed markedly lower induction levels after iron-ion exposure compared to equivalent doses of X rays. Quantification of pATF2 and pSMC1 foci revealed fewer cells containing foci and fewer foci per cell after iron-ion compared to X-ray exposure. These findings suggest that ATM responds to DSBs induced by high-LET radiation differently from DSBs induced by low-LET radiation.     

Involvement of the endoplasmic reticulum in peroxisome formation

The traditional view holds that peroxisomes are autonomous organelles multiplying by growth and division. More recently, new observations have challenged this concept. Herein, we present evidence supporting the involvement of the endoplasmic reticulum (ER) in peroxisome formation by electron microscopy, immunocytochemistry and three-dimensional image reconstruction of peroxisomes and associated compartments in mouse dendritic cells. We found the peroxisomal membrane protein Pex13p and the ATP-binding cassette transporter protein PMP70 present in specialized subdomains of the ER that were continuous with a peroxisomal reticulum from which mature peroxisomes arose. The matrix proteins catalase and thiolase were only detectable in the reticula and peroxisomes. Our results suggest the existence of a maturation pathway from the ER to peroxisomes and implicate the ER as a major source from which the peroxisomal membrane is derived.     

RAPID EVOLUTION OF REPRODUCTIVE ISOLATION BETWEEN INCIPIENT OUTCROSSING AND SELFING CLARKIA SPECIES

A major goal of speciation research is to understand the processes involved in the earliest stages of the evolution of reproductive isolation (RI). One important challenge has been to identify systems where lineages have very recently diverged and opportunities for hybridization are present. We conducted a comprehensive examination of the components of RI across the life cycle of two subspecies of Clarkia xantiana, which diverged recently (ca. 65,000 bp). One subspecies is primarily outcrossing, but self‐compatible, whereas the other is primarily selfing. The subspecies co‐occur in a zone of sympatry but hybrids are rarely observed. Premating barriers resulted in nearly complete isolation in both subspecies with flowering time and pollinator preference (for the outcrosser over the selfer) as the strongest barriers. We found that the outcrosser had consistently more competitive pollen, facilitating hybridization in one direction, but no evidence for pollen–pistil interactions as an isolating barrier. Surprisingly, postzygotic isolation was detected at the stage of hybrid seed development, but in no subsequent life stages. This crossing barrier was asymmetric with crosses from the selfer to outcrosser most frequently failing. Collectively, the results provide evidence for rapid evolution of multiple premating and postzygotic barriers despite a very recent divergence time.     

Invariant chain modulates HLA class II protein recycling and peptide presentation in nonprofessional antigen presenting cells

The expression of MHC class II molecules and the invariant chain (Ii) chaperone, is coordinately regulated in professional antigen presenting cells (APC). Ii facilitates class II subunit folding as well as transit and retention in mature endosomal compartments rich in antigenic peptides in these APC. Yet, in nonprofessional APC such as tumors, fibroblasts and endocrine tissues, the expression of class II subunits and Ii may be uncoupled. Studies of nonprofessional APC indicate class II molecules access antigenic peptides by distinct, but poorly defined pathways in the absence of Ii. Here, investigations demonstrate that nonprofessional APC such as human fibroblasts lacking Ii internalize antigenic peptides prior to the binding of these ligands to recycling class II molecules. By contrast, fibroblast lines expressing Ii favor exogenous peptides binding directly to cell surface class II molecules without a need for ligand internalization. Endocytosis of class II molecules was enhanced in cells lacking Ii compared with Ii-expressing APC. These results suggest enhanced reliance on the endocytic recycling pathway for functional class II presentation in nonprofessional APC.     

Synthesis and activity of small molecule GPR40 agonists

The first report on the identification and structure-activity relationships of a novel series of GPR40 agonists based on a 3-(4-{[N-alkyl]amino}phenyl)propanoic acid template is described. Structural modifications to the original screening hit yielded compounds with a 100-fold increase in potency at the human GPR40 receptor and pEC(50)s in the low nanomolar range. The carboxylic acid moiety is not critical for activity but typically elicits an agonistic response higher than those observed with carboxamide replacements. These compounds may prove useful in unraveling the therapeutic potential of this receptor for the treatment of Type 2 diabetes.     

Changes in the secretion and glycosylation of fibronectin by human skin fibroblasts associated with tuberous sclerosis

Fibroblasts from skin and skin lesions of patients with tuberous sclerosis (TS) and from skin of normal individuals were grown in culture. ELISA showed that the spent medium of those derived from TS skin lesions contained significantly more fibronectin (FN) than spent medium from the other cells. Amino acid compositional analysis of the FN from TS and normal sources revealed no substantial differences. However the FN of fibroblasts from TS-skin lesions was shown by HPAEC to contain a two- to three-fold increased content of carbohydrate. The changed monosaccharide composition was consistent with an increased content of N- and O-linked glycans and with the former containing polylactosamine chains. Fibroblasts from a normal individual were shown to proliferate more slowly and to produce larger cells when grown on FN from a TS skin lesion compared to growth on FN from normal skin. This revised version was published online in November 2006 with corrections to the Cover Date.     

Parasitism and decreased response to sex pheromones in malePeriplaneta americana (Dictyoptera: Blattidae)

Parasites are known to alter the behavior of their hosts, but little is known about their effects on responses to sexual stimuli. Periplaneta americanainfected with the acanthocephalan Moniliformis moniliformiswere compared to uninfected animals in their behavioral and electroantennogram responses to a synthetic P. americanapheromone component, periplanone-B, and a pheromone mimic, bornyl acetate. In a t-maze there was no significant difference between infected animals' responses to periplanone-B and a random binomial distribution; the responses of uninfected animals were significantly nonrandom. The electroan-tennogram responses of infected and uninfected animals to bornyl acetate or periplanone-B did not differ significantly, however, indicating that the alteration probably does not occur at the peripheral level but at a central nervous system level.     

Inversions and other unusual heteromorphisms detected by C-banding

Summary Sixteen patients with unusual heteromorphisms involving alterations of the length and/or position of centromeric heterochromatin are described. Family studies showed that the heteromorphisms were present in other relatives and segregated in the expected 1:1 ratio. There was a significantly greater frequency of unusual heteromorphisms among Orientals than in other races studied.     

A Regional Multiple-Stressor Rank-Based Ecological Risk Assessment for the Fjord of Port Valdez,Alaska

We conducted an ecological risk assessment of the marine environment of Port Valdez, a fjord in south-central Alaska. Because the assessment was regional rather than site-specific and contained a large number of different stressors in a variety of environments, we required a nontraditional method to estimate risks. We created a Relative Risk Model to rank and sum individual risks numerically within each subarea, from each source, and to each habitat. Application of this model involved division of Port Valdez into 11 subareas containing specific ecological and anthropogenic structures and activities. Within each subarea, the stressor sources were analyzed to estimate exposure of receptors within habitats leading to effects relevant to the chosen assessment endpoints. The subareas were analyzed and compared to form a Port-wide perspective of ecological risk. Available chemical concentrations from sediment and mussels collected from the Port were compared to various toxicological benchmarks as a partial confirmation of the risk analysis. An estimation of the risk of polycyclic aromatic hydrocarbons (PAHs) to marine invertebrates indicated low risk. The municipal boat harbor had the highest estimate, which reflected our relative risk rankings. The Relative Risk Model approach appears robust and has potential for use in situations where multiple stressors are of concern and for assessments covering broad geographic areas. In the Port Valdez assessment the approach provided relative risk rankings for chemical and physical stressors from various sources. But data were available for confirmation of risk estimates only for the chemical stressors. The rankings are relative, and extrapolation beyond the scenario in which they were developed is not warranted. Uncertainty is large, and the numerical scores collapse a multidimensional space into a single value. Use of just the numerical score out of context is more valid than with other indexes. The value of the approach lies in the relative rankings and the accounting of the components of the relative risk score.