Dietary arsenic affects dimethylhydrazine-induced aberrant crypt formation and hepatic global DNA methylation and DNA methyltransferase activity in rats

Cell culture studies have suggested that arsenic exposure results in decreased S-adenosylmethionine (SAM), causing DNA hypomethylation. Previously, we have shown that hepatic SAM is decreased and/or S-adenosylhomocysteine increased in arsenic-deprived rats; these rats tended to have hypomethylated DNA. To determine, the effect of dietary arsenic on dimethylhydrazine (DMH)-induced aberrant crypt formation in the colon, Fisher 344 weanling male rats were fed diets containing 0,05, or 50 μg As (as NaAsO₂)/g. After 12 wk, dietary arsenic affected the number of aberrant crypts (p<0.02) and aberrant crypt foci (p<0.007) in the colon and the amount of global DNA methylation (p<0.04) and activity of DNA methyltransferase (DNMT) (p<0.003) in the liver. In each case, there were more aberrant crypts and aberrant crypt foci, a relative DNA hypomethylation, and increased activity of DNMT in the rats fed 50 μg As/g compared to those fed 0.5 μg As/g. The same phenomenon, an increased number of aberrant crypts and aberrant crypt foci, DNA hypomethylation, and increased DNMT tended to hold when comparing rats fed the diet containing no supplemental arsenic compared to rats fed 0.5 μg As/g. The data suggest that there is a threshold for As toxicity and that possibly too little dietary As could also be detrimental. The U.S. Department of Agriculture, Agricultural Research Service. Northern Plains Area is an equal opportunity/affirmative action employer and all agency services are available without discrimination.     

PAK5 kinase is an inhibitor of MARK/Par-1, which leads to stable microtubules and dynamic actin

MARK/Par-1 is a kinase involved in development of embryonic polarity. In neurons, MARK phosphorylates tau protein and causes its detachment from microtubules, the tracks of axonal transport. Because the target sites of MARK on tau occur at an early stage of Alzheimer neurodegeneration, we searched for interaction partners of MARK. Here we report that MARK2 is negatively regulated by PAK5, a neuronal member of the p21-activated kinase family. PAK5 suppresses the activity of MARK2 toward its target, tau protein. The inhibition requires the binding between the PAK5 and MARK2 catalytic domains, but does not require phosphorylation. In transfected Chinese hamster ovary (CHO) cells both kinases show a vesicular distribution with partial colocalization on endosomes containing AP-1/2. Although MARK2 transfected alone destabilizes microtubules and stabilizes actin stress fibers, PAK5 keeps microtubules stable through the down-regulation of MARK2 but destabilizes the F-actin network so that stress fibers and focal adhesions disappear and cells develop filopodia. The results point to an inverse relationship between actin- and microtubule-related signaling by the PAK5 and MARK2 pathways that affect both cytoskeletal networks.     

Brain aging: reorganizing discoveries about the aging mind

New discoveries challenge the long-held view that aging is characterized by progressive loss and decline. Evidence for functional reorganization, compensation and effective interventions holds promise for a more optimistic view of neurocognitive status in later life. Complexities associated with assigning function to age-specific activation patterns must be considered relative to performance and in light of pathological aging. New biological and genetic markers, coupled with advances in imaging technologies, are enabling more precise characterization of healthy aging. This interdisciplinary, cognitive neuroscience approach reveals dynamic and optimizing processes in aging that might be harnessed to foster the successful aging of the mind.     

Synaptic mitochondria are critical for mobilization of reserve pool vesicles at Drosophila neuromuscular junctions

In a forward screen for genes affecting neurotransmission in Drosophila, we identified mutations in dynamin-related protein (drp1). DRP1 is required for proper cellular distribution of mitochondria, and in mutant neurons, mitochondria are largely absent from synapses, thus providing a genetic tool to assess the role of mitochondria at synapses. Although resting Ca2+ is elevated at drp1 NMJs, basal synaptic properties are barely affected. However, during intense stimulation, mutants fail to maintain normal neurotransmission. Surprisingly, FM1-43 labeling indicates normal exo- and endocytosis, but a specific inability to mobilize reserve pool vesicles, which is partially rescued by exogenous ATP. Using a variety of drugs, we provide evidence that reserve pool recruitment depends on mitochondrial ATP production downstream of PKA signaling and that mitochondrial ATP limits myosin-propelled mobilization of reserve pool vesicles. Our data suggest a specific role for mitochondria in regulating synaptic strength.     

Abeta42 is essential for parenchymal and vascular amyloid deposition in mice

Considerable circumstantial evidence suggests that Abeta42 is the initiating molecule in Alzheimer's disease (AD) pathogenesis. However, the absolute requirement for Abeta42 for amyloid deposition has never been demonstrated in vivo. We have addressed this by developing transgenic models that express Abeta1-40 or Abeta1-42 in the absence of human amyloid beta protein precursor (APP) overexpression. Mice expressing high levels of Abeta1-40 do not develop overt amyloid pathology. In contrast, mice expressing lower levels of Abeta1-42 accumulate insoluble Abeta1-42 and develop compact amyloid plaques, congophilic amyloid angiopathy (CAA), and diffuse Abeta deposits. When mice expressing Abeta1-42 are crossed with mutant APP (Tg2576) mice, there is also a massive increase in amyloid deposition. These data establish that Abeta1-42 is essential for amyloid deposition in the parenchyma and also in vessels.     

Generation and characterization of pluripotent stem cells from cloned bovine embryos

Bovine embryonic stem (ES) cell lines reported to date vary in morphology and marker expression (e.g., alkaline phosphatase [ALPL], stage-specific embryonic antigen 4 [SSEA4], and OCT4) that normally are associated with the undifferentiated, pluripotent state. These observations suggest that the proper experimental conditions for consistently producing bovine ES cells have not been identified. Here, we report three bovine ES cell lines, one from in vitro-fertilized and two from nuclear transfer embryos. These bovine ES cells grew in large, multicellular colonies resembling the mouse ES and embryonic germ (EG) cells and human EG cells. Throughout the culture period, most of the cells within the colonies stained positive for ALPL and the cell surface markers SSEA4 and OCT4. The staining patterns of nuclear transfer ES cells were identical to those of the blastocysts generated in vitro yet different from most previously reported bovine ES cell lines, which were either negative or not detected. After undifferentiated culture for more than 1 yr, these cells maintained the ability to differentiate into embryoid bodies and derivatives of all three EG layers, thus demonstrating their pluripotency. However, unlike the mouse and human ES cells, following treatment with trypsin, type IV collagenase, or protease E, our bovine ES cells failed to self-renew and became spontaneously differentiated. Presumably, this resulted from an interruption of the self-renewal pathway. In summary, we generated pluripotent bovine ES cells with morphology similar to those of established ES cells in humans and mice as well as marker-staining patterns identical to those of the bovine blastocysts.     

Monoclonal Antibodies Against Soybean Bowman-Birk Inhibitor Recognize the Protease-Reactive Loops

Monoclonal antibodies against soybean Bowman-Birk protease inhibitor (BBI) have been generated and used to detect and quantify BBI in foods, soybean germplasm, and animal tissues and fluids. The purpose of this study was to determine the recognition sites of two monoclonal antibodies to BBI (mAb 238 and mAb 217) in relation to the protease-inhibitory sites of BBI. The results showed that (1) the binding of mAb 238 can be blocked by trypsin and that of mAb 217 by chymotrypsin; (2) the trypsin or chymotrypsin inhibitory activities of BBI are blocked by mAb 238 or mAb 217, respectively; and (3) mAb 238 failed to recognize a tryptic loop mutant BBI variant and mAb 217 was unable to bind a chymotryptic loop mutant BBI variant. These findings demonstrate that the epitopes recognized by mAb 238 and mAb 217 reside, at least in part, in the tryptic and chymotryptic loops of BBI, respectively.     

Magnesium promotes flagellation of Vibrio fischeri

The bacterium Vibrio fischeri requires bacterial motility to initiate colonization of the Hawaiian squid Euprymna scolopes. Once colonized, however, the bacterial population becomes largely unflagellated. To understand environmental influences on V. fischeri motility, we investigated migration of this organism in tryptone-based soft agar media supplemented with different salts. We found that optimal migration required divalent cations and, in particular, Mg2+. At concentrations naturally present in seawater, Mg2+ improved migration without altering the growth rate of the cells. Transmission electron microscopy and Western blot experiments suggested that Mg2+ addition enhanced flagellation, at least in part through an effect on the steady-state levels of flagellin protein.