The determination of allergen-specific IgE and IgG antibodies in patients sensitized to the house dust mite Dermatophagoides pteronyssinus

45 patients, hypersensitive to house-dust mites, were examined by the method of skin tests to D. pteronyssinus allergen. Besides, in their blood sera the levels of allergen-specific IgE antibodies were determined in the radioallergosorbent test and allergen-specific IgG antibodies, in the enzyme immunoassay. These tests revealed that in 91% of the patients the results of skin tests were positive, in 68% an elevated level of specific IgE antibodies and in 93% of the patients an elevated level of specific IgG antibodies were detected. All patients showed the positive result in one of the above-mentioned tests. The largest group of the patients (55%) included persons showing the positive result of the skin test and having elevated levels of allergen-specific IgG and IgE antibodies. Thus, in cases of hypersensitivity to house-dust mites the levels of allergen-specific IgG and IgE antibodies in the patients' blood sera should be determined.     

Predicting the population dynamics of the house dust mite Dermatophagoides pteronyssinus (Acari: Pyroglyphidae) in response to a constant hygrothermal environment using a model of the mite life cycle

A generalised model of the life cycle of a house dust mite, which can be tailored to any particular species of domestic mite, is presented. The model takes into account the effects of hygrothermal conditions on each life cycle phase. It is used in a computer simulation program, called POPMITE, which, by incorporating a population age structure, is able to predict population dynamics. The POPMITE simulation is adapted to the Dermatophagoides pteronyssinus (Acari: Pyroglyphidae) (DP) mite using published data on the egg development period, total development period, adult longevity, mortality during egg development, mortality during juvenile development, and fecundity of individual DP mites held at a range of constant hygrothermal conditions. An example is given which illustrates how the model functions under constant hygrothermal conditions. A preliminary validation of POPMITE is made by a comparison of the POPMITE predictions with published measurements of population growth of DP mites held at a range constant hygrothermal conditions for 21 days. The POPMITE simulation is used to provide predictions of population growth or decline for a wide range of constant relative humidity and temperature combinations for 30 and 60 days. The adaptation of the model to correctly take account of fluctuating hygrothermal conditions is discussed.     

Real‐time cell analysis and heat shock protein gene expression in the TcA Tribolium castaneum cell line in response to environmental stress conditions

The rust red flour beetle, Tribolium castaneum (Herbst, 1797) (Coleoptera: Tenebrionidae), is a pest of stored grain and one of the most studied insect model species. Some of the previous studies involved heat response studies in terms of survival and heat shock protein expression, which are regulated to protect other proteins against environmental stress conditions. In the present study, we characterize the impedance profile with the xCELLigence Real‐Time Cell Analyzer and study the effect of increased temperature in cell growth and viability in the cell line BCIRL‐TcA‐CLG1 (TcA) of T. castaneum. This novel system measures cells behavior in real time and is applied for the first time to insect cells. Additionally, cells are exposed to heat shock, increased salinity, acidic pH and UV‐A light with the aim of measuring the expression levels of Hsp27, Hsp68a, and Hsp83 genes. Results show a high thermotolerance of TcA in terms of cell growth and viability. This result is likely related to gene expression results in which a significant up‐regulation of all studied Hsp genes is observed after 1 h of exposure to 40 °C and UV light. All 3 genes show similar expression patterns, but Hsp27 seems to be the most affected. The results of this study validate the RTCA method and reveal the utility of insect cell lines, real‐time analysis and gene expression studies to better understand the physiological response of insect cells, with potential applications in different fields of biology such as conservation biology and pest management.     

Real-time PCR as an effective technique to assess the impact of phoresy by Paenibacillus sp. bacteria on Steinernema diaprepesi nematodes in nature

Quantitative real‐time PCR (qPCR) is a powerful tool to study species of cryptic organisms in complex food webs. This technique was recently developed to detect and quantify several species of entomopathogenic nematodes (EPNs), which are widely used for biological control of insects, and some natural enemies of EPNs such as nematophagous fungi and the phoretic bacteria Paenibacillus sp. and Paenibacillus nematophilus. A drawback to the use of primers and TaqMan probes designed for Paenibacillus sp. is that the qPCR also amplified Paenibacillus thiaminolyticus and Paenibacillus popilliae, two closely related species that are not phoretically associated with EPNs. Here, we report that the detection of Paenibacillus sp. DNA in nematode samples was two orders of magnitude greater (P < 0.001) when the bacterium was added to soil together with its EPN species‐specific host Steinernema diaprepesi than when it was added concomitantly with other EPNs or with species of bacterial‐feeding nematodes. Just 6% of samples detected trace amounts of P. thiaminolyticus and P. popilliae exposed to the same experimental conditions. Thus, although the molecular assay detects Paenibacillus spp. DNA in nonphoretic associations, the levels are essentially background compared to the detection of Paenibacillus sp. in association with its nematode host.     

Characterization of the complete porcine MSTN gene and expression levels in pig breeds differing in muscularity

Myostatin (MSTN), a transforming growth factor beta superfamily member, is an essential factor for the growth and development of muscle mass. The protein functions as a negative regulator of muscle growth and is related to the so-called double-muscling phenotype in cattle, where a series of mutations renders the gene inactive. One particular breed of pigs, the Belgian Piétrain, also shows a heavily muscled phenotype. The similarity of muscular phenotypes between the double-muscled cattle and Piétrain pigs indicated that MSTN may be a candidate gene for muscular hypertrophy in pigs. In this study, we sequenced and analysed the complete MSTN gene from 45 pigs of five different breeds, including the heavily muscled Piétrain breed at one extreme and the Meishan and Wild boar breeds at the other extreme. In total, 7626 bp of the porcine MSTN gene were sequenced, including the 5' and 3' UTR. Fifteen polymorphic loci were found, three of which were located in the promoter region, five in intron 1 and seven in intron 2. Most mutations were found when comparing the obtained MSTN sequence with porcine MSTN sequences already published. However, one polymorphism located at position 447 of the porcine MSTN promoter had a very high allele frequency in the Piétrain pig breed and disrupted a putative myocyte enhancer factor 3 binding site. Real-time PCR using Sybr Green showed that this mutation was associated with expression levels of the MSTN gene in m. longissimus dorsi at an age of 4 weeks.     

A transient expression assay for the in planta efficacy screening of an antimicrobial peptide against grapevine bacterial pathogens

Aims: Natural and synthetic antimicrobial peptides (AMPs) are of increasing interest as potential resistance conferring elements in plants against pathogen infection. The efficacy of AMPs against pathogens is prescreened by in vitro assays, and promising AMP candidates are introduced as transgenes into plants. As in vitro and in planta environments differ, a prescreening procedure of the AMP efficacy in the plant environment is desired. Here, we report the efficacy of the purified synthetic peptide D4E1 against the grapevine‐infecting bacterial pathogens Agrobacterium vitis and Xylophilus ampelinus in vitro and describe for the first time an in planta prescreening procedure based on transiently expressed D4E1. Methods and Results: The antimicrobial effect of D4E1 against Ag. vitis and X. ampelinus was shown by a reduction in colony‐forming units in vitro in a traditional plate‐based assay and by a reduction in bacterial titres in planta as measured by quantitative real‐time PCR (qPCR) in grapevine leaves transiently expressing D4E1. A statistically significant reduction in titre was shown for X. ampelinus, but for Ag. vitis, a significant reduction in titre was only observed in a subset of plants. Conclusions: The titres of both grapevine‐infecting bacterial pathogens were reduced in an in vitro assay and for X. ampelinus in an in planta assay by D4E1 application. This widens the applicability of D4E1 as a potential resistance‐enhancing element to additional pathogens and in a novel plant species. Significance and Impact of the Study: D4E1 is a promising candidate to confer enhanced resistance against the two tested grapevine bacterial pathogens, and the applied transient expression system proved to be a valuable tool for prescreening of D4E1 efficacy in an in planta environment. The described prescreening procedure can be used for other AMPs and might be adapted to other plant species and pathogens before the expensive and tedious development of stably transgenic lines is started.     

不同胁迫条件下化感与非化感水稻PAL多基因家族的差异表达

水稻苯丙氨酸解氨酶(PAL)调控酚酸类化感物质的合成代谢。编码PAL的基因是一个基因家族,包含至少11个基因成员,并受不同环境条件的调控。为了明确PAL基因家族中调控水稻化感作用的特定基因成员,本研究运用实时荧光定量PCR技术分析了低氮及稗草胁迫条件下强化感水稻PI312777与非化感水稻Lemont中根系的11个PAL成员基因的表达差异。结果表明,低氮和稗草胁迫条件下,PI312777和Lemont中的 PAL4和PAL10均不表达,其余9个PAL基因成员发生了不同程度的表达变化。其中,PAL11均上调表达,其分别在低氮处理和稗草胁迫的PI312777中上调3.29倍和1.07倍,而在相同处理下的Lemont中上调3.92倍和1.08倍;PAL3和PAL9则仅在低氮和稗草胁迫条件下的PI312777中上调表达,低氮胁迫分别为1.83倍和2.66倍,稗草胁迫为1.46倍和2.65倍;而这两个基因在相同处理下的Lemont中表达下调,低氮胁迫下调1.05和1.24倍,稗草胁迫下调1.14和1.16倍,推测PAL3和PAL9可能与胁迫初期调控水稻化感作用有关。     

Cloning and differential expression of the growth hormone in Pampus argenteus

The growth hormone (GH) is a pluripotent hormone produced by the pituitary in vertebrates. It plays important roles in the growth, development, and metabolism of vertebrates.We cloned GH cDNA sequence of Pampus argenteus (GenBank: KT257176). Multi‐sequence analysis revealed P. argenteus GH cDNA contained four conservative cysteine residues positions (Cys⁶⁹, Cys¹⁷⁷, Cys¹⁹⁴, and Cys²⁰²) and shared more than 51.5% identity with homologues from other reported bony fish GHs, except that of Lepisosteus osseus. We used semi‐quantitative RT‐PCR and quantitative real‐time PCR to detect GH expression in 10 tissues and GH expression levels in the pituitary at six different growth stages, and also detected GH content in serum at different growth stages . qPCR showed that GH mRNA was detected in the liver, muscle, kidney, intestine, pituitary, olfactory bulb, stomach, heart, gill, and ovary. The highest level of P. argenteus GH mRNA was observed in the pituitary (P < 0.01, n = 3). At different growth stages, P. argenteus GH expression first increased, decreased, and increased again. GH gene expression levels and the variations of serum GH levels of P. argenteus were consistent with the growth rate and associated with the sexual maturity. In addition, in situ hybridization was used to locate the GH expression in pituitary. In situ hybridization showed that the GH‐positive cells were round, oval, or irregular and often gathered into groups or presented branches along the nerve fibers.