A simple treatment to significantly increase signal specificity in immunohistochemistry

It is increasingly important to understand a gene’s function at the protein level. Immunohistochemistry is used to identify new functions of phytohormones by finding their precise localization patterns. To facilitate protein immunolocalization, we developed a protocol that can significantly reduce background and give highly specific signals of antibody to traget proteins. The key improvement is to treat secondary antibodies by mixing with homogenized fresh and fixed plant tissue. Using this protocol, we have successfully carried out immunolocalization of proteins encoded by genesAP3 andHoMADS2, as well as plant hormones ABA and IAA. These authors contributed equally     

High volume hydrodynamic injection of plasmid DNA via the hepatic artery results in a high level of gene expression in rat hepatocellular carcinoma induced by diethylnitrosamine

BACKGROUND: Hydrodynamic injection of naked plasmid DNA (pDNA) via the tail vein is a safe and effective method of gene transfer to the liver. However, successful gene transfer has yet to be shown for hepatocellular carcinoma (HCC); therefore, we investigated the feasibility and efficacy of hydrodynamic injection via the tail vein and hepatic artery in a diethylnitrosamine (DEN)-induced HCC model in rats. METHODS: HCC was induced in Sprague-Dawley rats by 100 ppm DEN in drinking water. pCMV-SPORT-beta-galactosidase (beta-gal, 400 microg) was injected (i) via the tail vein in a volume of 0.1 ml/g in 30 s or (ii) via the hepatic artery in a volume of 5 or 10 ml at 1 ml/s, either with or without temporary occlusion of the inferior vena cava (IVC) and portal vein (PV). The liver was harvested 24 h after administration, and beta-gal expression was evaluated with X-gal staining and measurement of enzymatic activity in tissue homogenates. RESULTS: Hydrodynamic injection via the tail vein achieved transgene expression only in non-cancerous tissue (tumor: 0.16 +/- 0.04%, non-tumor: 5.07 +/- 1.66%). Hydrodynamic injection via the hepatic artery was tolerated, but failed to produce efficient transgene expression in tumor and non-tumor cells. On the other hand, concomitant use of temporary IVC/PV occlusion with hydrodynamic injection via the hepatic artery dramatically increased transgene expression in cancer cells, but tumor-selective gene transfer was not achieved with this procedure (tumor: 7.38 +/- 3.66%, non-tumor: 7.77 +/- 1.06%). CONCLUSIONS: High-volume hydrodynamic injection of a pDNA solution via the hepatic artery with IVC/PV occlusion achieved a high level of gene expression in a HCC rat model. This gene transfer technique may have potential in clinical gene therapy for HCC.     

Attenuation of mouse acute colitis by naked hepatocyte growth factor gene transfer into the liver

BACKGROUND: Hepatocyte growth factor (HGF) has multiple biological effects on a wide variety of cells. It modulates intestinal epithelial proliferation and migration, and critically regulates intestinal wound healing. AIMS: To investigate the therapeutic effect of HGF gene transfer, we introduced the HGF gene into the liver of mice with acute colitis. METHODS: The rat HGF expression plasmid vector, pCAGGS-HGF, was injected via the tail vein into C57BL/6 mice, followed by dosing with dextran sulfate sodium in distilled water. Firstly, the HGF gene was injected once on day 0. Secondly, the HGF gene was injected on day 0 and again on day 2. RESULTS: Injection of the HGF gene ameliorated colitis with inhibition of both loss of body weight and shortening of colon length. It protected the colon from epithelial erosions and cellular infiltration. Expression of mRNAs for IFN-gamma, IL18, and TNF-alpha was reduced in the colon. In contrast, expression of mRNA for IL-10 was increased. The numbers of BrdU-positive intestinal epithelial cells were increased, and the numbers of TUNEL-positive apoptotic cells were decreased. Furthermore, a second injection prolonged the elevation of serum HGF levels, and ameliorated the symptoms better than a single injection. The empty pCAGGS plasmid did not ameliorate acute colitis. CONCLUSIONS: HGF gene transfer attenuated acute colitis by facilitating intestinal wound repair as well as inhibiting inflammation, suggesting a new strategy for treatment of IBD.     

Mechanism of plasmid delivery by hydrodynamic tail vein injection. II. Morphological studies

BACKGROUND: The efficient delivery of plasmid DNA (pDNA) to hepatocytes by a hydrodynamic tail vein (HTV) procedure has greatly popularized the use of naked nucleic acids. The hydrodynamic process renders onto the tissue increased physical forces in terms of increased pressures and shear forces that could lead to transient or permanent membrane damage. It can also trigger a series of cellular events to seal or reorganize the stretched membrane. Our goal was to study the uptake mechanism by following the morphological changes in the liver and correlate these with the fate of the injected plasmid DNA. METHODS: We utilized both light microscopic (LM) and electron microscopic (EM) techniques to determine the effect of the HTV procedure on hepatocytes and non-parenchymal cells at various times after injection. The LM studies used paraffin-embedded livers with hematoxylin and eosin (H&E) staining. The immune-EM studies used antibodies labeled with sub-nanometer gold particles followed by silver enhancement to identify the location of injected pDNA at the subcellular level. The level of overall damage to liver cells was estimated based on alanine aminotransferase (ALT) release and clearance. RESULTS: Both the LM and EM results showed the appearance of large vesicles in hepatocytes as early as 5 min post-injection. The number of vesicles decreased by 20-60 min. Plasmid DNA molecules often appeared to be associated with or inside such vesicles. DNA could also be detected in the space of Disse, in the cytoplasm and in nuclei. Non-parenchymal cells also contained DNA, but HTV-induced vesicles could not be observed in them. CONCLUSIONS: Our studies suggest an alternative or additional pathway for naked DNA into hepatocytes besides direct entry via membrane pores. It may be difficult to prove which of these pathways lead to gene expression, but the membrane pore hypothesis alone appears insufficient to explain why expression happens preferentially in hepatocytes. Further study is needed to delineate the importance of each of these putative pathways and their interrelationship in enabling oligonucleotide (siRNA) activity and pDNA expression.     

Mutagenesis and subsite mapping underpin the importance for substrate specificity of the aglycon subsites of glycoside hydrolase family 11 xylanases

Glycoside hydrolase family (GH) 11 xylanase A from Bacillus subtilis (BsXynA) was subjected to site-directed mutagenesis to probe the role of aglycon active site residues with regard to activity, binding of decorated substrates and hydrolysis product profile. Targets were those amino acids identified to be important by 3D structure analysis of BsXynA in complex with substrate bound in the glycon subsites and the + 1 aglycon subsite. Several aromatic residues in the aglycon subsites that make strong substrate–protein interactions and that are indispensable for enzyme activity, were also important for the specificity of the xylanase. In the + 2 subsite of BsXynA, Tyr65 and Trp129 were identified as residues that are involved in the binding of decorated substrates. Most interestingly, replacement of Tyr88 by Ala in the + 3 subsite created an enzyme able to produce a wider variety of hydrolysis products than wild type BsXynA. The contribution of the + 3 subsite to the substrate specificity of BsXynA was established more in detail by mapping the enzyme binding site of the wild type xylanase and mutant Y88A with labelled xylo-oligosaccharides. Also, the length of the cord – a long loop flanking the aglycon subsites of GH11 xylanases – proved to impact the hydrolytic action of BsXynA. The aglycon side of the active site cleft of BsXynA, therefore, offers great potential for engineering and design of xylanases with a desired specificity.     

Developmental duration as an organizer of the evolving mammalian brain: scaling,adaptations, and exceptions

Neurodevelopmental duration plays a central role in the evolution of the retina and neocortex in mammals. In the diurnal primate eye and retina, it is necessary to scale the number of cones versus the number of rods with different exponents to defend their respective functions of spatial acuity and sensitivity in eyes of different sizes. The order of photoreceptor precursor specification, cones specified first, rods second, couples their respective cell numbers at maturity to the kinetics of embryonic stem cell proliferation. Different durations of retinogenesis change the ratio of rods to cones produced so as to defend both functions over a range of eye diameters. In the evolution of nocturnality, the same coupling of photoreceptor specification to neurogenesis is altered to fewer cones and many more rods in nocturnal eyes, by delaying the onset of retinogenesis. Similarly, the neocortex also shows coupling of the specification of laminar position with duration of neurogenesis. Overall, duration of neurogenesis directly predicts neocortex volume in most mammalian clades. In larger brains with longer neocortical neurogenesis, its organization changes progressively, differentiating the frontal pole from the occipital pole in volume of connectivity and number of neurons per unit column. This permits greater, hierarchically organized information abstraction with increasing neocortex volume. Exceptions do exist, however, in species of three separate taxa, marsupials, naked mole rats, and bats, which break the correlation of neurodevelopmental duration and brain size. Naked mole rats and bats both have small brains and unusual longevity, coupled with neurodevelopmental periods characteristic of much bigger‐brained animals, raising the possibility that developmental duration and lifespan have some genetic or mechanistic control in common. The role of duration of development in mediating between the mechanistic levels of construction of retinal and cortical organization, and the different life histories associated with larger brains, such as duration of parental care, learning and overall longevity are discussed.     

Barley yellow dwarf virus transmission and feeding behaviour of Rhopalosiphum padi on Hordeum bulbosum clones

A Hordeum bulbosum L. (Poaceae) clone A17 was identified, which showed complete resistance to Barley yellow dwarf virus (BYDV) and Cereal yellow dwarf virus (CYDV). It was not possible to infect plants of A17 with BYDV‐PAV, ‐MAV, or with CYDV‐RPV by the aphid vectors Rhopalosiphum padi (L.) or Sitobion avenae (Fabricius) (both Hemiptera: Aphididae). Plants of the A17 clone and of the BYDV‐susceptible H. bulbosum clone A21 revealed some resistance to R. padi compared to the susceptible winter barley cultivar Rubina [Hordeum vulgare L. (Poaceae)]. The development time to the imago was longer and the number of nymphs was reduced on both clones compared with cv. Rubina. The probing and feeding behaviour of R. padi on plants of the H. bulbosum clones was studied over 12 h and compared with that on plants of the barley cv. Rubina. Principal component analysis of the results of the feeding behaviour revealed a clear separation of the H. bulbosum genotypes from Rubina. On H. bulbosum the number of penetrations was higher but total feeding time was shorter. Significant differences were mainly found in the phloem feeding parameters for plants of both clones in comparison to Rubina, with the virus resistant A17 clone having the strongest effect and the susceptible A21 clone being intermediate. Most significant differences were found in parameters of the phloem salivation phase. On A17, an average of less than one (0.9) E1 phase per plant was observed (3.3 on A21 and 5.7 on Rubina) and its duration was reduced to less than 1 min (0.9 min) in comparison to 2.4 min on A21 and 5.7 min on Rubina. Also, the phloem feeding (E2) phase was clearly reduced on A17 plants with 0.5 E2 phases per test and a mean duration of 1.1 min in contrast with 2.9 and 3.5 E2 phases per test and 34.1 and 421.3 min for A21 and Rubina, respectively. These results point towards a phloem‐localized factor for aphid resistance in H. bulbosum, i.e., on A17 plants the phloem salivation time is too short for a successful infection by BYDV leading to vector resistance.     

结瘤因子和苏芸金菌素对干旱胁迫下燕麦产量及其保护酶活性的影响

结瘤因子(LCO)和苏芸金菌素(TH17)在逆境环境下对作物生长具有重要调节作用。该试验在盆栽条件下,以‘坝莜3号’燕麦品种为材料,分别在苗期、拔节期和抽穗期进行干旱胁迫并叶面喷施浓度为0.01 μmol/L的LCO和TH17,比较燕麦产量、叶片渗透调节物质和几种保护酶活性的变化情况。结果显示:(1)与喷施清水对照（CK）相比,正常水分条件下喷施LCO和TH17能促进燕麦生长,提高产量;在干旱胁迫下(SS)喷施LCO和TH17能减轻燕麦株高、穗长、穗粒数、生物产量、单株粒重以及干物质积累速率的降低幅度,且在苗期喷施LCO和TH17燕麦单穗粒重增加幅度最高,分别较干旱胁迫对照（SS_CK）提高61.25%和65.00%。(2)干旱胁迫下,LCO和TH17使游离脯氨酸含量增加最多的时期是拔节期,分别较SS_CK增加27.19%和41.00%;可溶性总糖在抽穗期提高幅度最大,分别较SS_CK高61.43%和112.54%;可溶性蛋白在苗期提高幅度最大,且分别较SS_CK提高9.58%和13.80%。(3)干旱胁迫下,LCO和TH17使SOD和POD活性增加幅度最大的时期均是抽穗期,CAT活性则是在拔节期增加幅度最大。(4)干旱胁迫下,在苗期LCO和TH17使燕麦叶片相对电导率和MDA含量降低幅度最大,分别较SS_CK降低17.61%、36.67%和43.43%、30.28%。研究表明,在苗期喷施LCO和TH17使燕麦产量提高幅度最高;干旱条件下,不同时期喷施LCO和TH17能提高燕麦自身渗透调节能力、抗氧化保护酶活性和增强细胞膜的稳定性,从而使其更好地适应干旱条件。     

Characterisation of cDNAs for ribulose bisphosphate carboxylase small subunit genes in different species of Avena (Poaceae)

ABSTRACT

We report the cloning and sequencing of 14 rbcS cDNAs in six species of Avena (Poaceae) with different genome and ploidy levels. The nucleotide sequences 504 bp long were aligned with the published sequences of cultivated hexaploid oat, wheat, barley, rye, rice and corn and subjected to cladistic and phenetic analyses. The parsimonious analysis generated a tree with a topology very similar to the phenogram generated by the Neighbor-Joining analysis based on the Jukes and Cantor distances. Within the monophyletic assemblage of the tribe Aveneae, consistent clades composed of rbcS clones belonging to different species are recognized. It is suggested that they correspond to orthologous genes belonging to different subfamilies, and that the “within-species?d homogenisation may have occurred at a slow rate with respect to species evolution. In the monophyletic group of Pooideae, the topologies place barley rbcS sequences closer to wheat and rye than to oat sequences. This grouping agrees with most taxonomic and phylogenetic views.