Microspore embryogenesis and programmed cell death in barley: effects of copper on albinism in recalcitrant cultivars

Albinism remains a major problem in cereal improvement programs that rely on doubled haploid (DH) technology, and the factors controlling the phenomenon are not well understood. Here we report on the positive influence of copper on the production of DH plants obtained through microspore embryogenesis (ME) in recalcitrant cultivars of barley (Hordeum vulgare L.). The presence of copper sulphate in the anther pre-treatment medium improved green DH plant regeneration from cultivars known to produce exclusively albino plants using classical procedures. In plastids, the effect of copper was characterized by a decrease in starch and a parallel increase in internal membranes. The addition of copper sulphate in the ME pre-treatment medium should enable breeders to exploit the genetic diversity of recalcitrant cultivars through DH technology. We examined programmed cell death (PCD) during microspore development to determine whether PCD may interfere with the induction of ME and/or the occurrence of albinism. By examining the fate of nuclei in various anther cell layers, we demonstrated that the kinetics of PCD in anthers differed between the barley cultivars Igri and Cork that show a low and a high rate of albinism, respectively. However, no direct correlation between PCD in the anther cell layers and the rate of albinism was observed and copper had no influence on the PCD kinetic in these cultivars. It was concluded that albinism following ME was not due to PCD in anthers, but rather to another unknown phenomenon that appears to specifically affect plastids during microspore/pollen development.     

The level of BMP4 signaling is critical for the regulation of distinct T-box gene expression domains and growth along the dorso-ventral axis of the optic cup

Background  

Polarised gene expression is thought to lead to the graded distribution of signaling molecules providing a patterning mechanism across the embryonic eye. Bone morphogenetic protein 4 (Bmp4) is expressed in the dorsal optic vesicle as it transforms into the optic cup. Bmp4 deletions in human and mouse result in failure of eye development, but little attempt has been made to investigate mammalian targets of BMP4 signaling. In chick, retroviral gene overexpression studies indicate that Bmp4 activates the dorsally expressed Tbx5 gene, which represses ventrally expressed cVax. It is not known whether the Tbx5 related genes, Tbx2 and Tbx3, are BMP4 targets in the mammalian retina and whether BMP4 acts at a distance from its site of expression. Although it is established that Drosophila Dpp (homologue of vertebrate Bmp4) acts as a morphogen, there is little evidence that BMP4 gradients are interpreted to create domains of BMP4 target gene expression in the mouse.     

Nest desertion by a cowbird host: an antiparasite behavior or a response to egg loss?

Natural selection can favor songbirds that desert nests containingeggs of the parasitic brown-headed cowbird (Molothrus ater).However, the high variability in desertion of parasitized nestswithin species is perplexing in light of the typically highcosts of parasitism. Because nest desertion can also be a responseto partial clutch predation, we first asked if Bell's vireos(Vireo bellii) deserted nests in response to the presence ofcowbird eggs (antiparasite response hypothesis) or to egg removalby predators and female cowbirds (egg predation hypothesis).Second, we asked whether variation in nest desertion was dueto intrinsic differences among individuals or to variation innest contents. We monitored a large number of nests (n = 494)and performed a clutch manipulation experiment to test thesehypotheses. The number of vireo eggs that remained in a nestwas a strong predictor of desertion both within and among pairs.Neither the presence of a single cowbird egg, which leads tonest failure for this host, nor the number of cowbird eggs receivedin a vireo nest influenced nest desertion. Furthermore, vireosdid not desert experimental nests when we immediately exchangedcowbird eggs for vireo eggs but deserted if we removed vireoeggs and replaced them with cowbird eggs the following morning.Desertion of parasitized nests by Bell's vireos can be almostentirely explained as a response to partial or complete clutchloss and does not appear to have been altered by selection frombrood parasitism.     

Upregulated miR-146a expression in peripheral blood mononuclear cells from rheumatoid arthritis patients

Introduction

MicroRNAs are small noncoding RNA molecules that negatively regulate gene expression via degradation or translational repression of their targeted mRNAs. It is known that aberrant microRNA expression can play important roles in cancer, but the role of microRNAs in autoimmune diseases is only beginning to emerge. In this study, the expression of selected microRNAs is examined in rheumatoid arthritis.

Methods

Total RNA was isolated from peripheral blood mononuclear cells obtained from patients with rheumatoid arthritis, and healthy and disease control individuals, and the expression of miR-146a, miR-155, miR-132, miR-16, and microRNA let-7a was analyzed using quantitative real-time PCR.

Results

Rheumatoid arthritis peripheral blood mononuclear cells exhibited between 1.8-fold and 2.6-fold increases in miR-146a, miR-155, miR-132, and miR-16 expression, whereas let-7a expression was not significantly different compared with healthy control individuals. In addition, two targets of miR-146a, namely tumor necrosis factor receptor-associated factor 6 (TRAF6) and IL-1 receptor-associated kinase 1 (IRAK-1), were similarly expressed between rheumatoid arthritis patients and control individuals, despite increased expression of miR-146a in patients with rheumatoid arthritis. Repression of TRAF6 and/or IRAK-1 in THP-1 cells resulted in up to an 86% reduction in tumor necrosis factor-α production, implicating that normal miR-146a function is critical for the regulation of tumor necrosis factor-α production.

Conclusions

Recent studies have shown that synovial tissue and synovial fibroblasts from patients with rheumatoid arthritis exhibit increased expression of certain microRNAs. Our data thus demonstrate that microRNA expression in rheumatoid arthritis peripheral blood mononuclear cells mimics that of synovial tissue/fibroblasts. The increased microRNA expression in rheumatoid arthritis patients is potentially useful as a marker for disease diagnosis, progression, or treatment efficacy, but this will require confirmation using a large and well defined cohort. Our data also suggest a possible mechanism contributing to rheumatoid arthritis pathogenesis, whereby miR-146a expression is increased but unable to properly function, leading to prolonged tumor necrosis factor-α production in patients with rheumatoid arthritis.     

Giantin is the major Golgi autoantigen in human anti-Golgi complex sera

Anti-Golgi complex antibodies (AGAs) are primarily associated with systemic lupus erythematosus and Sjögren's syndrome. Here we report on the immunoreactivity of AGAs against five Golgi autoantigens (giantin, golgin-245, golgin-160, golgin-95/GM130, and golgin-97) and provide data from epitope mapping on the most common Golgi autoantigen, namely giantin. A total of 80 human sera containing AGAs, as defined by indirect immunofluorescence on HEp-2 cells, were analyzed by ELISA using recombinant autoantigens and immunoprecipitation. The proportion of AGA sera that reacted with the five Golgi autoantigens was correlated with the molecular mass of the Golgi antigens. Autoantibodies to giantin, the largest Golgi autoantigen, were the predominant AGAs, being found in 50% of the AGA sera. Epitope mapping of giantin was performed using six recombinant fragments spanning the entire protein. Antigiantin-positive sera with low titer autoantibodies recognized epitopes in the carboxyl-terminal fragments that are proximal to the Golgi membrane, whereas higher titer sera exhibited strong reactivity to amino-terminal and central domains that are likely to extend from the Golgi membrane into the cytoplasm. Our working hypothesis is that aberrantly expressed Golgi complex autoantigens may be released into the immune system when cells undergo lysis. By virtue of a carboxyl-terminal transmembrane domain, giantin is likely to be more stably associated with the cytoplasmic face of the Golgi complex than are other golgins, which are peripheral proteins. The stable association of giantin with the putative released Golgi complex may contribute to its preferential autoantigenicity.     

Familial transmission of the FMR1 CGG repeat.

To better define the nature of FMR1 CGG-repeat expansions, changes in allele sizes for 191 families with fragile X and for 33 families with gray-zone repeats (40-60) were analyzed. Expansion of the fragile X chromosome to the full mutation was seen in 13.4% of offspring from premutation mothers with 56-59 repeats, 20.6% of those with 60-69 repeats, 57.8% of those with 70-79 repeats, 72.9% of those with 80-89 repeats, and 97.3% of those with 90-199 repeats. For premutation fathers, the majority (62%) of their daughters had a larger repeat number, while a few had either a smaller (22%) or the same (16%) repeat number, compared with their fathers' sizes. However, daughters with a smaller repeat number were observed only if their fathers had > or = 80 repeats. Fifteen (39.5%) of 38 such daughters carried a smaller repeat than did their fathers. We observed that a similar repeat number was inherited more often than expected by chance, among the members of a sibship segregating fragile X. This familial clustering, observed in the offspring of both males and females with a premutation, implies there may be an additional factor, independent of parental repeat size, that influences CGG-repeat instability. Instability in gray-zone allele transmissions was observed in 25% of alleles with 50-60 CGGs but in <8% of those with 40-49 CGGs. Examination of gray-zone allele organization revealed that long tracts of pure CGGs (>34) are not always unstably transmitted. These results raise new questions regarding the familial factors that may determine transmission expansions.     

Long-term treatment of severe hypertension with minoxidil.

Minoxidil, a new potent hypotensive agent, was used as the primary antihypertensive agent in 11 patients--10 men and 1 woman aged 35 to 54 years with severe hypertension that was refractory to treatment with maximal (or maximally tolerated) doses of conventional antihypertensive agents. Six patients had severely impaired renal function and three of them were undergoing long-term hemodialysis. The patients were given 2.5 to 40 mg/d of minoxidil for periods of 2 to 29 months. All except one who was almost anuric received propranolol and diuretics. Blood pressure was controlled satisfactorily in all patients. In two patients the hypertension became partially resistant after 1 year of treatment. The main side effects were sodium retention, tachycardia and hirsutism. Renal function remained stable or improved and hemodialysis was discontinued in two patients. Minoxidil is a remarkably potent hypotensive with relatively few side effects and seems particularly advantageous in patients with chronic renal failure.     

Simultaneous determination of total homocysteine, cysteine, cysteinylglycine, and glutathione in human plasma by high-performance liquid chromatography: application to studies of oxidative stress

A sensitive, reproducible, and robust high-performance liquid chromatography (HPLC) method has been validated for simultaneously determining total concentrations of the aminothiols homocysteine, cysteine, cysteinylglycine, and glutathione in human plasma. Plasma aminothiols are reduced via incubation with tris-(2-carboxyethyl)-phosphine hydrochloride, followed by protein precipitation with trichloroacetic acid and derivatization with ammonium-7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonic acid. Separation of aminothiols and the internal standard mercaptopropionylglycine is achieved using reversed-phase HPLC conditions and fluorescence detection. Excellent linearity is observed for all analytes over their respective concentration ranges with correlation coefficients (r) > 0.99. The intra- and inter-day precision and accuracy were within +/-10%. This method utilizes an internal standard, employs phosphate buffered saline-based standards and quality controls, and demonstrates excellent plasma recovery and improved sensitivity. This assay is well suited for high-throughput quantitative determination of aminothiols in clinical studies, and is currently being used to support investigations of oxidative stress in patients with chronic kidney disease.