Genetic mapping of quantitative trait loci affecting susceptibility to Marek's disease virus induced tumors in F2 intercross chickens.

Marek''s disease (MD) is a lymphoproliferative disease caused by the MD virus (MDV), which costs the poultry industry nearly $1 billion annually. To identify quantitative trait loci (QTL) affecting MD susceptibility, the inbred lines 6(3) (MD resistant) and 7(2) (MD susceptible) were mated to create more than 300 F2 chickens. The F2 chickens were challenged with MDV JM strain, moderately virulent) at 1 wk of age and assessed for MD susceptibility. The QTL analysis was divided into three stages. In stage 1, 65 DNA markers selected from the chicken genetic maps were typed on the 40 most MD-susceptible and the 40 most MD-resistant F2 chickens, and 21 markers residing near suggestive QTL were revealed by analysis of variance (ANOVA). In stage 2, the suggestive markers plus available flanking markers were typed on 272 F2 chickens, and three suggestive QTL were identified by ANOVA. In stage 3, using the interval mapping program Map Manager and permutation tests, two significant and two suggestive MD QTL were identified on four chromosomal subregions. Three to five loci collected explained between 11 and 23% of the phenotypic MD variation, or 32-68% of the genetic variance. This study constitutes the first report in the domestic chicken on the mapping of non-major histocompatibility complex QTL affecting MD susceptibility.     

HO-1-mediated macroautophagy: a mechanism for unregulated iron deposition in aging and degenerating neural tissues

Oxidative stress, deposition of non-transferrin iron, and mitochondrial insufficiency occur in the brains of patients with Alzheimer disease (AD) and Parkinson disease (PD). We previously demonstrated that heme oxygenase-1 (HO-1) is up-regulated in AD and PD brain and promotes the accumulation of non-transferrin iron in astroglial mitochondria. Herein, dynamic secondary ion mass spectrometry (SIMS) and other techniques were employed to ascertain (i) the impact of HO-1 over-expression on astroglial mitochondrial morphology in vitro , (ii) the topography of aberrant iron sequestration in astrocytes over-expressing HO-1, and (iii) the role of iron regulatory proteins (IRP) in HO-1-mediated iron deposition. Astroglial hHO-1 over-expression induced cytoplasmic vacuolation, mitochondrial membrane damage, and macroautophagy. HO-1 promoted trapping of redox-active iron and sulfur within many cytopathological profiles without impacting ferroportin, transferrin receptor, ferritin, and IRP2 protein levels or IRP1 activity. Thus, HO-1 activity promotes mitochondrial macroautophagy and sequestration of redox-active iron in astroglia independently of classical iron mobilization pathways. Glial HO-1 may be a rational therapeutic target in AD, PD, and other human CNS conditions characterized by the unregulated deposition of brain iron.     

Cloning of plant cDNAs encoding calmodulin-binding proteins using35S-labeled recombinant calmodulin as a probe

Radiolabelled calmodulin has previously been used to screen cDNA expression libraries to isolate calmodulin-binding proteins. We have modified this technique for the isolation of plant calmodulin-binding proteins. [³⁵S]-methionine was used instead of the inorganic [³⁵S]-sulfate, or¹²⁵I used in previous methods. In addition, theE. coli pET expression system was chosen to obtain high levels of recombinant calmodulin at the time of labelling. The procedure thus takes into account both the specific activity of the probe and the amount of protein necessary for screening a large number of filters. Here we describe in detail a procedure for the production and purification of [³⁵S]-recombinant calmodulin and the use of the radiolabelled protein as a probe to screen plant cDNA expression libraries. The [³⁵S]-labeled calmodulin probe easily detects the λICM-1 phage encoding a partial mouse calmodulin-dependent protein kinase II that was previously isolated using a [¹²⁵I]-calmodulin probe (Sikela and Hahn, 1987). Subsequently, a tobacco root cDNA expression library was screened and a positive clone encoding a calcium-dependent calmodulin-binding protein was isolated.     

Cardiovascular Disease Risk of Abdominal Obesity vs. Metabolic Abnormalities

It remains unclear whether abdominal obesity increases cardiovascular disease (CVD) risk independent of the metabolic abnormalities that often accompany it. Therefore, the objective of this study was to evaluate the independent effects of abdominal obesity vs. metabolic syndrome and diabetes on the risk for incident coronary heart disease (CHD) and stroke. The Framingham Offspring, Atherosclerosis Risk in Communities, and Cardiovascular Health studies were pooled to assess the independent effects of abdominal obesity (waist circumference >102 cm for men and >88 cm for women) vs. metabolic syndrome (excluding the waist circumference criterion) and diabetes on risk for incident CHD and stroke in 20,298 men and women aged ≥45 years. The average follow‐up was 8.3 (s.d. 1.9) years. There were 1,766 CVD events. After adjustment for demographic factors, smoking, alcohol intake, number of metabolic syndrome components, and diabetes, abdominal obesity was not significantly associated with an increased risk of CVD (hazard ratio (HR) (95% confidence interval): 1.09 (0.98, 1.20)). However, after adjustment for demographics, smoking, alcohol intake, and abdominal obesity, having 1–2 metabolic syndrome components, the metabolic syndrome and diabetes were each associated with a significantly increased risk of CVD (2.12 (1.80, 2.50), 2.82 (1.92, 4.12), and 5.33 (3.37, 8.41), respectively). Although abdominal obesity is an important clinical tool for identification of individuals likely to possess metabolic abnormalities, these data suggest that the metabolic syndrome and diabetes are considerably more important prognostic indicators of CVD risk.     

Mechanical reconfiguration mediates swallowing and rejection in Aplysia californica

Muscular hydrostats, such as tongues, trunks or tentacles, have fewer constraints on their degrees of freedom than musculoskeletal systems, so changes in a structure’s shape may alter the positions and lengths of other components (i.e., induce mechanical reconfiguration). We studied mechanical reconfiguration during rejection and swallowing in the marine mollusk Aplysia californica. During rejection, inedible material is pushed out of an animal’s buccal cavity. The grasper (radula/odontophore) closes on inedible material, and then a posterior muscle, I2, pushes the grasper toward the jaws (protracts it). After the material is released, an anterior muscle complex (the I1/I3/jaw complex) pushes the grasper toward the esophagus (retracts it). During swallowing, the grasper is protracted open, and then retracts closed, pulling in food. Grasper closure changes its shape. Magnetic resonance images show that grasper closure lengthens I2. A kinetic model quantified the changes in the ability of I2 and I1/I3 to exert force as grasper shape changed. Grasper closure increases I2’s ability to protract during rejection, and increases I1/I3’s ability to retract during swallowing. Motor neurons controlling radular closure may therefore affect the behavioral outputs of I2’s and I1/I3’s motor neurons. Thus, motor neurons may modulate the outputs of other motor neurons through mechanical reconfiguration.Valerie A. Novakovic and Gregory P. Sutton contributed equally to the paper.     

Synchronized infection of cell cultures by magnetically controlled virus

To override the diffusion-limited adsorption step of viral infection, we magnetically synchronized cell attachment. Human immunodeficiency virus type 1-based lentivirus preparations were rendered magnetically reactive by association with magnetite nanoparticles, 50 nm in diameter. Application of a magnetic field resulted in immediate redistribution of the viral inoculum to the cell-associated state and completion of the productive adsorption process within 1 min. Independent of adsorption time, viral concentration, and diffusion rate, infection subsequently progressed by the receptor-mediated entry mechanism. Synchronization of this rate-limiting step of infection may now be applied to analyze isolated events in the viral replication sequence.     

Cellular immunity to Coccidioides immitis: In vitro lymphocyte response to spherules,arthrospores, and endospores

In vitro stimulation of incorporation of tritiated thymidine by human peripheral lymphocytes in response to two soluble antigens and three different intact but nonviable fungal forms of Coccidioides immitis was studied. Lymphocytes were obtained from three groups of subjects: healthy skin test positive, healthy skin test negative, and disseminated disease. Dose-response relationships to the intact forms (endospores, arthrospores, and spherules) were determined. Responses of lymphocytes from healthy skin test-positive subjects and subjects with disseminated disease were similar. Ranking of antigens by “potency” gave the following results: endospores = spherulin > mycelial filtrate > arthrospores = spherules. Endospores were the most potent of the intact forms in 10 of 11 subjects. The clear superiority of endospores over spherules is not due to differences in the total particle surface area available for presentation to the leukocytes. All antigens tested except spherules could discriminate between skin test-positive and skin test-negative subjects in this in vitro system. A T-cell-enriched, B-cell- and mono-cyte-depleted cell population demonstrated an active response to spherulin and to endospores. The variance of these finding with animal studies demonstrating spherules to be immunogenically superior when compared to endospores is discussed. This may have importance in future studies in humans of vaccines to C. immitis.     

Activation Energy for Glucose-Induced Germination of Bacillus megaterium Spores

The maximum germination rate of Bacillus megaterium QM B1551 spores in glucose increased, and the lag before its attainment decreased, with increasing germination temperature. The activation energy for germination (μ = approximately 20 kcal/mole), based on rate or on lag, was consistent with an enzymatic mechanism.