Phosphorescence quenching microrespirometry of skeletal muscle in situ

We have developed an optical method for the evaluation of the oxygen consumption (Vo(2)) in microscopic volumes of spinotrapezius muscle. Using phosphorescence quenching microscopy (PQM) for the measurement of interstitial Po(2), together with rapid pneumatic compression of the organ, we recorded the oxygen disappearance curve (ODC) in the muscle of the anesthetized rats. A 0.6-mm diameter area in the tissue, preloaded with the phosphorescent oxygen probe, was excited once a second by a 532-nm Q-switched laser with pulse duration of 15 ns. Each of the evoked phosphorescence decays was analyzed to obtain a sequence of Po(2) values that constituted the ODC. Following flow arrest and tissue compression, the interstitial Po(2) decreased rapidly and the initial slope of the ODC was used to calculate the Vo(2). Special analysis of instrumental factors affecting the ODC was performed, and the resulting model was used for evaluation of Vo(2). The calculation was based on the observation of only a small amount of residual blood in the tissue after compression. The contribution of oxygen photoconsumption by PQM and oxygen inflow from external sources was evaluated in specially designed tests. The average oxygen consumption of the rat spinotrapezius muscle was Vo(2) = 123.4 ± 13.4 (SE) nl O(2)/cm(3) · s (N = 38, within 6 muscles) at a baseline interstitial Po(2) of 50.8 ± 2.9 mmHg. This technique has opened the opportunity for monitoring respiration rates in microscopic volumes of functioning skeletal muscle.     

Inhibition of the Unfolded Protein Response by metformin in renal proximal tubular epithelial cells

All-trans retinoic acid (ATRA) induces cellular senescence via up-regulation of p16 and p21; however, the action mechanism of ATRA is unknown. Here, we show that ATRA induces promoter hypomethylation of p16 and p21 via down-regulation of DNA methyltransferases 1, 3a, and 3b to facilitate binding of Ets1/2 to the p16 promoter and p53 to the p21 promoter, resulting in up-regulation of their expression and subsequent induction of cellular senescence in HepG2 cells. These effects were mediated by retinoic acid receptor β₂ whose promoter was also hypomethylated in the presence of ATRA. Therefore, ATRA can be considered as an epi-drug in cancer therapy.     

Real-time PCR to determine transgene copy number and to quantitate the biolocalization of adoptively transferred cells from EGFP-transgenic mice

Quantitative real-time PCR (qPCR) is a sensitive technique for the detection and quantitation of specific DNA sequences. Here we describe a Taqman qPCR assay for quantification of tissue-localized, adoptively transferred enhanced green fluorescent protein (EGFP)-transgenic cells. A standard curve constructed from serial dilutions of a plasmid containing the EGFP transgene was (i) highly reproducible, (ii) detected as few as two copies, and (iii) was included in each qPCR assay. qPCR analysis of genomic DNA was used to determine transgene copy number in several mouse strains. Fluorescent microscopy of tissue sections showed that adoptively transferred vascular endothelial cells (VEC) from EGFP-transgenic mice specifically localized to tissue with metastatic tumors in syngeneic recipients. VEC microscopic enumeration of liver metastases strongly correlated with qPCR analysis of identical sections (Pearson correlation 0.81). EGFP was undetectable in tissue from control mice by qPCR. In another study using intra-tumor EGFP-VEC delivery to subcutaneous tumors, manual cell count and qPCR analysis of alternating sections also strongly correlated (Pearson correlation 0.82). Confocal microscopy of the subcutaneous tumor sections determined that visual fluorescent signals were frequently tissue artifacts. This qPCR methodology offers specific, objective, and rapid quantitation, uncomplicated by tissue autofluorescence, and should be readily transferable to other in vivo models to quantitate the biolocalization of transplanted cells.     

ECA3, a Golgi-localized P2A-type ATPase, plays a crucial role in manganese nutrition in Arabidopsis

Calcium (Ca) and manganese (Mn) are essential nutrients required for normal plant growth and development, and transport processes play a key role in regulating their cellular levels. Arabidopsis (Arabidopsis thaliana) contains four P(2A)-type ATPase genes, AtECA1 to AtECA4, which are expressed in all major organs of Arabidopsis. To elucidate the physiological role of AtECA2 and AtECA3 in Arabidopsis, several independent T-DNA insertion mutant alleles were isolated. When grown on medium lacking Mn, eca3 mutants, but not eca2 mutants, displayed a striking difference from wild-type plants. After approximately 8 to 9 d on this medium, eca3 mutants became chlorotic, and root and shoot growth were strongly inhibited compared to wild-type plants. These severe deficiency symptoms were suppressed by low levels of Mn, indicating a crucial role for ECA3 in Mn nutrition in Arabidopsis. eca3 mutants were also more sensitive than wild-type plants and eca2 mutants on medium lacking Ca; however, the differences were not so striking because in this case all plants were severely affected. ECA3 partially restored the growth defect on high Mn of the yeast (Saccharomyces cerevisiae) pmr1 mutant, which is defective in a Golgi Ca/Mn pump (PMR1), and the yeast K616 mutant (Deltapmc1 Deltapmr1 Deltacnb1), defective in Golgi and vacuolar Ca/Mn pumps. ECA3 also rescued the growth defect of K616 on low Ca. Promoter:beta-glucuronidase studies show that ECA3 is expressed in a range of tissues and cells, including primary root tips, root vascular tissue, hydathodes, and guard cells. When transiently expressed in Nicotiana tabacum, an ECA3-yellow fluorescent protein fusion protein showed overlapping expression with the Golgi protein GONST1. We propose that ECA3 is important for Mn and Ca homeostasis, possibly functioning in the transport of these ions into the Golgi. ECA3 is the first P-type ATPase to be identified in plants that is required under Mn-deficient conditions.     

Amylin causes anorexigenic effects via the hypothalamus and brain stem in chicks

This study was conducted to determine the effects of amylin on appetite-related processes in chicks. Broiler chicks were centrally and peripherally injected with amylin, and feed and water intake were quantified. Feed intake was reduced after both central and peripheral amylin, but water intake was not affected. To determine if the hypothalamus and brainstem were involved in the anorexigenic effect, chicks were centrally and peripherally injected with amylin, and c-Fos immunoreactivity was quantified in the lateral hypothalamus (LH), ventromedial hypothalamus (VMH), area postrema (AP) and the nucleus of the solitary tract (NTS). Amylin decreased c-Fos immunoreactivity in the LH, did not affect the VMH, and increased c-Fos immunoreactivity in the AP and NTS. To determine if alimentary transit time was affected, chicks received central amylin and were gavaged with chicken feed slurry containing a visible marker. Amylin-treated chicks had increased alimentary canal transit time. Chicks also responded to central amylin with increased anxiety-related behaviors and increased plasma corticosterone concentration. These results demonstrate that amylin affects feeding, alimentary canal transit, and behavior through hypothalamic and brainstem mechanisms in chicks.     

Pathfinding in a large vertebrate axon tract: isotypic interactions guide retinotectal axons at multiple choice points

Navigating axons respond to environmental guidance signals, but can also follow axons that have gone before - pioneer axons. Pioneers have been studied extensively in simple systems, but the role of axon-axon interactions remains largely unexplored in large vertebrate axon tracts, where cohorts of identical axons could potentially use isotypic interactions to guide each other through multiple choice points. Furthermore, the relative importance of axon-axon interactions compared with axon-autonomous receptor function has not been assessed. Here, we test the role of axon-axon interactions in retinotectal development, by devising a technique to selectively remove or replace early-born retinal ganglion cells (RGCs). We find that early RGCs are both necessary and sufficient for later axons to exit the eye. Furthermore, introducing misrouted axons by transplantation reveals that guidance from eye to tectum relies heavily on interactions between axons, including both pioneer-follower and community effects. We conclude that axon-axon interactions and ligand-receptor signaling have co-equal roles, cooperating to ensure the fidelity of axon guidance in developing vertebrate tracts.     

Multiple common susceptibility variants near BMP pathway loci GREM1, BMP4, and BMP2 explain part of the missing heritability of colorectal cancer

Genome-wide association studies (GWAS) have identified 14 tagging single nucleotide polymorphisms (tagSNPs) that are associated with the risk of colorectal cancer (CRC), and several of these tagSNPs are near bone morphogenetic protein (BMP) pathway loci. The penalty of multiple testing implicit in GWAS increases the attraction of complementary approaches for disease gene discovery, including candidate gene- or pathway-based analyses. The strongest candidate loci for additional predisposition SNPs are arguably those already known both to have functional relevance and to be involved in disease risk. To investigate this proposition, we searched for novel CRC susceptibility variants close to the BMP pathway genes GREM1 (15q13.3), BMP4 (14q22.2), and BMP2 (20p12.3) using sample sets totalling 24,910 CRC cases and 26,275 controls. We identified new, independent CRC predisposition SNPs close to BMP4 (rs1957636, P = 3.93×10(-10)) and BMP2 (rs4813802, P = 4.65×10(-11)). Near GREM1, we found using fine-mapping that the previously-identified association between tagSNP rs4779584 and CRC actually resulted from two independent signals represented by rs16969681 (P = 5.33×10(-8)) and rs11632715 (P = 2.30×10(-10)). As low-penetrance predisposition variants become harder to identify-owing to small effect sizes and/or low risk allele frequencies-approaches based on informed candidate gene selection may become increasingly attractive. Our data emphasise that genetic fine-mapping studies can deconvolute associations that have arisen owing to independent correlation of a tagSNP with more than one functional SNP, thus explaining some of the apparently missing heritability of common diseases.     

Seroprevalence of Trypanosoma cruzi in raccoons from South Carolina and Georgia

Trypanosoma cruzi, the causative agent of American trypanosomiasis or Chagas' disease, is of both medical and veterinary importance as is evidenced by chronic phase myocarditis in humans and dogs. Further, T. cruzi has been reported from over 20 species of wildlife reservoir hosts in the USA, with raccoons (Procyon lotor) and opossums (Didelphis virginiana) being the most common. Whereas previous studies on T. cruzi in raccoons have included only culture and direct examination of blood, the indirect immunofluorescent antibody test (IFAT) was used in the current study to detect anti-T. cruzi antibodies in the serum of raccoons. Of 221 raccoons trapped at 13 sites representing the five physiographic regions of South Carolina plus five sites in the Piedmont region of Georgia (from April 1997 to February 2000), 104 (47%) were seropositive. A higher seroprevalence in raccoons was observed in the coastal regions, with seroprevalence in the Lower Coastal Plain South (61%) being significantly higher than that in the Foothills (37%), Piedmont (42%), and Upper Coastal Plain (40%) regions. However, at a seroprevalence of 52%, the Lower Coastal Plain North was not significantly different from any other region. Although more female raccoons were infected than males, no statistical difference in prevalence was observed between sexes. The high seroprevalence of T. cruzi in raccoons, together with a few reports of wildlife isolates being infective for other wildlife species and domestic/laboratory animals, suggests that risk of T. cruzi infection may be higher than previously suspected.     

Dietary NaCl influences the organization of chorda tympani neurons projecting to the nucleus of the solitary tract in rats

Prior research has shown that maintained exposure to either a low or high NaCl diet from conception to adulthood is associated with changes in NaCl solution intake and neural responses of the chorda tympani (CT) nerve. The present study examined the influence of maintained exposure to a low or high NaCl diet on the central organization of CT neurons projecting to the nucleus of the solitary tract (NST). Three groups of rats were reared and maintained on regular chow containing either basal 0.1%, intermediate 1.0% or high 6% NaCl from conception to adulthood. The fluorescent marker Dil was applied to the CT for characterization of afferent terminations and efferent cell body labeling in the brainstem. The total NST area occupied by CT afferent fibers was the same for all three dietary groups. However, the pattern of CT innervation differed such that there was an enlarged dorsal terminal field in the high group. There were no group differences in body and brain weight, or in efferent labeled neurons. Thus, Dil has been demonstrated to be an effective transport marker of the gustatory system and the parameters of dietary NaCl exposure that influence the pattern of the CT fibers projecting to the NST have been further clarified.     

Understanding dorsoventral topography: backwards and forwards

Retinal axons project to their central targets along two orthogonal topographic axes, anterior-posterior (A-P) and dorsal-ventral (D-V). While ephrin-A/EphA signaling determines A-P topography, little has been known about the molecular mechanisms guiding axons along the D-V axis. Two papers by Mann et al. and Hindges et al. in this issue of Neuron provide evidence for both forward and reverse ephrin-B/EphB signaling in regulating D-V topography.