Localization of zein genes in maize

The band patterns of zein polypeptides were determined for many commercial inbred corn lines and maize stocks using isofocusing in agarose gels and sodium dodecyl sulfate (SDS)-urea gels. Each inbred line or homozygous maize strain genotype has a distinct zein profile which has been catalogued according to the distance of charge migration and molecular weight (kilodaltons). Several zein polypeptides were mapped to chromosomes 4 and 10 with the use of reciprocal translocations. The mapping of at least two polypeptides on distal 4L and 10L had not been previously reported. The general methods used in the present research will permit the mapping of all the zein polypeptides to chromosomal sites.Pioneer Hi-Bred International provided financial support.     

Effects of lighting environment on the degeneration of retinal ganglion cells in glutamate/aspartate transporter deficient mice,a mouse model of normal tension glaucoma

Lighting conditions may affect the development of retinal degenerative diseases such as macular degeneration. In this study, to determine whether the lighting environment affects the progression of degeneration of retinal ganglion cells (RGCs), we examined glutamate/aspartate transporter (GLAST) heterozygous (GLAST^+/-) mice, a mouse model of normal tension glaucoma. GLAST^+/- mice were reared under a 12-h light-dark cycle (Light/Dark) or complete darkness (Dark/Dark) condition after birth. The total RGC number in the Dark/Dark group was significantly decreased compared with the Light/Dark group at 3 weeks old, while the number of osteopontin-positive αRGCs were similar in both groups. At 6 and 12 weeks old, the total RGC number were not significantly different in both conditions. In addition, the retinal function examined by multifocal electroretinogram were similar at 12 weeks old. These results suggest that lighting conditions may regulate the progression of RGC degeneration in some types of glaucoma.     

Alpha-lipoic acid affects the oxidative stress in various brain structures in mice with methionine and choline deficiency

Deficiency in methionine or choline can induce oxidative stress in various organs such as liver, kidney, heart, and brain. This study was to examine the effects of alpha-lipoic acid (LA) on oxidative stress induced by methionine and choline deficiency (MCD) in several brain structures. Male mice C57BL/6 (n = 28) were divided into four groups: (1) control – continuously fed with standard chow; (2) LA – fed with standard chow and receiving LA; (3) MCD2 – fed with MCD diet for two weeks, and (4) MCD2+LA – fed with MCD diet for two weeks and receiving LA (100 mg/kg/day intraperitonealy [i.p.]). Brain tissue (cortex, hypothalamus, striatum and hippocampus) was taken for determination of oxidative stress parameters. MCD diet induced a significant increase in malondialdehyde and NOx concentration in all brain regions, while LA restored their content to normal values. Similar to this, in MCD2 group, activity of total SOD, MnSOD, and Cu/ZnSOD was reduced by MCD diet, while LA treatment improved their activities in all brain structures. Besides, in MCD2 group a decrease in catalase activity in cortex and GSH content in hypothalamus was evident, while LA treatment induced an increase in catalase activity in cortex and striatum and GSH content in hypothalamus. LA treatment can significantly reduce lipid peroxidation and nitrosative stress, caused by MCD diet, in all brain regions by restoring antioxidant enzymes activities, predominantly total SOD, MnSOD, and Cu/ZnSOD, and to a lesser extent by modulating catalase activity and GSH content. LA supplementation may be used in order to prevent brain oxidative injury induced by methionine and choline deficiency.     

The cardiac repair benefits of inflammation do not persist: evidence from mast cell implantation

Multiple mechanisms contribute to progressive cardiac dysfunction after myocardial infarction (MI) and inflammation is an important mediator. Mast cells (MCs) trigger inflammation after MI by releasing bio‐active factors that contribute to healing. c‐Kit‐deficient (Kit^W/W‐v) mice have dysfunctional MCs and develop severe ventricular dilatation post‐MI. We explored the role of MCs in post‐MI repair. Mouse wild‐type (WT) and Kit^W/W‐v MCs were obtained from bone marrow (BM). MC effects on fibroblasts were examined in vitro by proliferation and gel contraction assays. MCs were implanted into infarcted mouse hearts and their effects were evaluated using molecular, cellular and cardiac functional analyses. In contrast to WT, Kit^W/W‐v MC transplantation into Kit^W/W‐v mice did not improve cardiac function or scar size post‐MI. Kit^W/W‐v MCs induced significantly reduced fibroblast proliferation and contraction compared to WT MCs. MC influence on fibroblast proliferation was Basic fibroblast growth factor (bFGF)‐dependent and MC‐induced fibroblast contractility functioned through transforming growth factor (TGF)‐β. WT MCs transiently rescue cardiac function early post‐MI, but the benefits of BM cell implantation lasted longer. MCs induced increased inflammation compared to the BM‐injected mice, with increased neutrophil infiltration and infarct tumour necrosis factor‐α (TNF‐α) concentration. This augmented inflammation was followed by increased angiogenesis and myofibroblast formation and reduced scar size at early time‐points. Similar to the functional data, these beneficial effects were transient, largely vanishing by day 28. Dysfunctional Kit^W/W‐v MCs were unable to rescue cardiac function post‐MI. WT MC implantation transiently enhanced angiogenesis and cardiac function. These data suggest that increased inflammation is beneficial to cardiac repair, but these effects are not persistent.     

UV‐absorbing films and nets affect the dispersal of western flower thrips,Frankliniella occidentalis (Thysanoptera: Thripidae)

UV‐absorbing films and nets are frequently used as covering materials for netted greenhouses and film tunnels in protected cultivation systems. This study explored the effects of such materials on the dispersal behaviour of western flower thrips (WFT), Frankliniella occidentalis, in flight cages under greenhouse conditions with additional artificial UV‐A light sources. The study involved release–recapture experiments in choice and no‐choice layouts. Different trapping methods were compared (blue sticky cards, plants and transparent cards) for recapture of thrips. In choice experiments, insects were released from a black box compartment between two tunnels covered with either UV‐transmitting or UV‐absorbing materials. A significantly higher proportion of (82–98%) WFT was recaptured in UV‐transmitting tunnels compared with UV‐absorbing tunnels. In no‐choice experiments, WFT were found to infiltrate the tunnels at different rates depending on the trap type used and experimental layout. In small‐scale dispersal experiments using blue sticky cards and plants as traps, infiltration was not significantly different between UV‐absorbing and UV‐transmitting tunnels, whereas when using transparent cards, WFT penetrated further into the UV‐transmitting plastic film tunnels. In larger‐scale dispersal experiments, plants or blue sticky cards were arranged in concentric circles around a source plant at the release point. Dispersal was found to differ depending on the method of release, but WFT tended to exhibit reduced dispersal from source plants under UV‐deficient conditions. In conclusion, our data support the hypothesis that manipulation of spectral light properties using UV‐absorbing cladding materials for protected crop stands interferes with the orientation and host finding of WFT, resulting in reduced dispersal into and within plant stands in UV‐deficient environments.     

Physical restriction of pods causes seed size reduction of a brassinosteroid-deficient faba bean (Vicia faba)

BACKGROUND AND AIMS: A brassinosteroid-deficient mutant faba bean (Vicia faba 'Rinrei') shows dwarfism in many organs including pods and seeds. 'Rinrei' has normal-sized seeds together with dwarf seeds, suggesting that dwarfism in the seed may be indirectly caused by brassinosteroid deficiency. The mechanism of seed size reduction in this mutant was investigated. METHODS: The associations between seed orientation in the pod, seed numbers per pod and pod lengths with seed sizes were analysed in 'Rinrei' and the wild-type plant. KEY RESULTS: 'Rinrei' seeds are tightly arranged in pods containing two or three seeds. Seed size decreased as the number of seeds per pod increased or as the length of the pod decreased. Where no physical restriction occurred between seeds in a pod, the wild-type faba bean seeds had a nearly constant size regardless of seed number per pod or pod length. 'Rinrei' seeds in pods containing single seeds were the same size as wild-type seeds. Brassinolide treatment increased the seed size and the length of pods containing three seeds in 'Rinrei'. CONCLUSION: Seed size of 'Rinrei' is mainly regulated through a reduction of pod length due to brassinosteroid deficiency; physical restriction within pods causes a reduction in seed size. These results suggest a possible mechanism for increasing faba bean yields to optimal levels.     

Downregulation of miR-122 in the rodent and human hepatocellular carcinomas

MicroRNAs (miRs) are conserved small non-coding RNAs that negatively regulate gene expression. The miR profiles are markedly altered in cancers and some of them have a causal role in tumorigenesis. Here, we report changes in miR expression profile in hepatocellular carcinomas (HCCs) developed in male Fisher rats-fed folic acid, methionine, and choline-deficient (FMD) diet. Comparison of the miR profile by microarray analysis showed altered expression of some miRs in hepatomas compared to the livers from age-matched rats on the normal diet. While let-7a, miR-21, miR-23, miR-130, miR-190, and miR-17-92 family of genes was upregulated, miR-122, an abundant liver-specific miR, was downregulated in the tumors. The decrease in hepatic miR-122 was a tumor-specific event because it did not occur in the rats switched to the folate and methyl-adequate diet after 36 weeks on deficient diet, which did not lead to hepatocarcinogenesis. miR-122 was also silent in a transplanted rat hepatoma. Extrapolation of this study to human primary HCCs revealed that miR-122 expression was significantly (P = 0.013) reduced in 10 out of 20 tumors compared to the pair-matched control tissues. These findings suggest that the downregulation of miR-122 is associated with hepatocarcinogenesis and could be a potential biomarker for liver cancers.     

A phosphorylation-independent role for the yeast cyclin-dependent kinase activating kinase Cak1

Cdc28 is the main cyclin-dependent kinase (CDK) directing the cell cycle in the budding yeast Saccharomyces cerevisiae. Besides cyclin binding, Cdc28 requires phosphorylation by the Cak1 kinase to achieve full activity. We have previously isolated carboxy-terminal cdc28^CST mutants that are temperature sensitive and exhibit high chromosome instability. Both phenotypes are suppressed by high copy Cak1 in a manner that is independent of its catalytic activity and conversely, combination of cdc28^CST and cak1 mutations results in synthetic lethality. Altogether, these results suggest that for the Cdc28 complexes to remain stable and active, an interaction with Cak1 is needed via the carboxyl terminus of Cdc28. We report two-hybrid assay data that support this model, and results that indicate that actively growing yeast cells require an optimum Cdc28:Cak1 ratio. While Cak1 is constitutively active and expressed, dividing cells tightly regulate Cak1 protein levels to ensure presence of adequate levels of Cdc28 CDK activity.     

Fas activation in adipocytes impairs insulin-stimulated glucose uptake by reducing Akt

Fas (CD95) belongs to the superfamily of the tumor necrosis factor (TNF) receptors. Besides its key role in apoptosis, Fas contributes to non-apoptotic pathways such as cell proliferation and inflammation. In 3T3-L1 adipocytes, activation of Fas by Fas ligand decreased insulin-stimulated glucose uptake, without affecting cell viability. This decrease in glucose uptake was accompanied by reduced protein expression and diminished phosphorylation of Akt. Similarly, insulin-stimulated glucose incorporation and protein levels of Akt were increased in isolated adipocytes from Fas deficient mice when compared to wild-type mice. In conclusion, Fas activation in adipocytes decreases Akt expression and thereby impairs insulin sensitivity.     

Construction of an inducible, pfkA and pfkB deficient strain of Escherichia coli for the expression and purification of phosphofructokinase from bacterial sources

As the key obligatory step in the glycolytic pathway, the regulation of phosphofructokinase (PFK-1) has been the focus of study of several laboratories. While standard cloning procedures have opened the door to study PFK from a vast array of sources, a good pfk knockout Escherichia coli strain has not previously been developed. Many laboratories rely on DF1020 or similar derivatives for PFK expression. Unfortunately, DF1020 grows poorly and does not have an inherent means for controlling expression of genes from plasmids. More importantly, however, DF1020 has a tendency to grow on minimal media when glucose is used as the sole carbon source. In this study, a new E. coli PFK expression strain lacking both PFK-1 and PFK-2 has been engineered using lambda-red mediated chromosomal deletion. The resulting strain has been designated RL257. In addition to having both pfkA and pfkB deleted, RL257 contains the lacI(q) allele, which allows for inducible expression when coupled with an expression vector containing either the lac or tac promoter.