Cloning and expression analysis of a muscarinic cholinergic receptor from the brain of ant,Polyrhachis vicina

Muscarinic acetylcholine receptors (mAchRs) are the predominant cholinergic receptors in the central and peripheral nervous systems of animals. They also have been found in various insect nervous systems. In this article, a full‐length cDNA of a pupative mAchR (PmAchR) was obtained from the brains of ant Polyrhachis vicina by homology cloning in combination with rapid amplification of cDNA ends. PmAchR encodes a 599‐amino acid protein that exhibits a high degree of homology with other mAchRs. Real‐time quantitative RT‐PCR analysis showed that PmAchR is differentially expressed in the brains of workers, males, and females. By in situ hybridization, it is revealed that PmAchR is widely expressed in different soma clusters of the brain, including the mushroom bodies, the antennal lobes, as well as the optic lobes (OL), and the most intensely staining is found in Kenyon cells. Nonetheless, there are more positive nerve fibers in the OL of males' brains than in females' and workers' brains. © 2011 Wiley Periodicals, Inc.     

Molecular modelling study of 2-phenylethynyladenosine (PEAdo) derivatives as highly selective A3 adenosine receptor ligands

A series of 2-phenylethynyladenosine (PEAdo) derivatives substituted in the N⁶- and 4′-position was synthesised and the new derivatives were tested at the four human adenosine receptors stably transfected into Chinese hamster ovary (CHO) cells, using radioligand binding studies (A₁, A_2A, A₃) or adenylyl cyclase activity assay (A_2B). Binding studies showed that the presence of a phenyl ethynyl group in the 2 position of adenosine favoured the interaction with A₃ receptors, resulting in compounds endowed with high affinity and selectivity for the A₃ subtype. Additional substitution of the N⁶- and 4′-position increases both A₃ affinity and selectivity. The results showed that the new compounds have a good affinity for the A₃ receptor and in particular, the N⁶-methoxy-2-phenylethynyl-5′-N-methylcarboxamidoadenosine, with a K_i at A₃ of 1.9 nM and a selectivity A₁/A₃ and A_2A/A₃ of 4,800- and 8,600-fold, respectively. Therefore, it is one of the most potent and selective agonists at the human A₃ adenosine receptor subtype reported so far. Furthermore, functional assays of inhibition of 10 μM forskolin-stimulated cAMP production via the adenosine A₃ receptor revealed that the new trisubstituted adenosine derivatives behave as full agonist of this receptor subtype. Docking analysis of these compounds was performed at a homology model of the human A₃ receptor based on the bovine rhodopsin crystal structure as template, and the results are in accordance with the biological data.An erratum to this article can be found at     

The diagnostic and prognostic role of MiRNA 15b and MiRNA 378a in neonatal sepsis

Background and objectivesSepsis is one of the major factors for both term and preterm babies with morbidity and mortality. On the basis of recent clinical trials, altered circulating micro-RNAs (miRNAs) may serve as possible biomarkers in sepsis for diagnosis and prognosis. The aim of this research is to assess the diagnostic and prognostic biomarkers of miRNA 15b and miRNA 378a for neonatal sepsis.Subjects & methodsThis study was carried out 25 neonates with sepsis admitted to neonatal ICU of Menoufia University Hospital and 25 healthy controls from February 2019 to May 2020. The relative quantification (RQ) of miRNA-15b and miRNA-378a expression was assessed using real time PCR technique. Results: Our results demonstrated that patients with sepsis had significantly higher level of MiRNA-15b than the healthy volunteers. On the other hand, patients with sepsis had significantly lower level of MiRNA-378a than the healthy volunteers. The ROC curve showed that the serum MiRNA-15b was a significant discriminator of sepsis with a combined sensitivity and specificity of 76% and 88% with cutoff point of 3.24. In addition, serum MiRNA-378a was a significant discriminator of sepsis with a combined sensitivity and specificity of 60% and 88% with cutoff point of 0.361. The miRNA-15b expression significantly correlated positive with respiratory rate (r =0.415,p =0.039), WBCs (r = 0.408, p =0.043), and CRP (r =0.407, p=0.043). Likewise, miRNA-378a expression significantly correlated negative with respiratory rate (r =-0.415p =0.024), WBCs (r =- 0.442, p =0.027), and CRP (r =- 0.459, p=0.021).ConclusionBoth MiRNA 15b and 378a are promising biomarker for neonatal sepsis.     

泛耐药细菌NDM-1基困Taqman PCR快速检测方法的建立

产NDM-1(New Delhi Metallo-β-lactamase 1,Ⅰ型新德里金属β-内酰胺酶)细菌是新近报道的一种泛耐药细菌,由于对绝大多数常用抗生素均耐药,又被称为超级细菌.目的:建立一种可快速检测泛耐药细菌NDM-1基因的Taqman探针实时荧光定量PCR法.方法:根据NDM-1基因序列,设计引物和Ta...     

Structure and function of invertebrate 5-HT receptors: a review

Over the last decade, knowledge of invertebrate serotonin receptors has expanded greatly. The first 5-HT receptor from Drosophila was cloned 10 years ago, and subsequently, 11 additional receptor genes have been cloned from Drosophila, molluscs (Lymnaea and Aplysia) and nematodes (Caenorhabditis and Ascaris). Information has also accumulated from physiological and biochemical studies that have used vertebrate serotonergic ligands to characterize endogenous invertebrate receptors. Although the endogenous receptors are often classified according to mammalian-based categories, in many cases the pharmacological properties of vertebrate and invertebrate receptors differ significantly and the actual identity of the latter is questionable. By providing information on the gene structure and amino acid sequence, molecular cloning studies offer a more definitive way to identify and classify invertebrate 5-HT receptors. This review summarizes information on the pharmacological and transductional properties of cloned invertebrate 5-HT receptors, and considers recent studies of endogenous receptors in the light of this new data.     

Molecular characterization of two nicotinic acetylcholine receptor subunits from Liposcelis bostrychophila Badonnel (Psocoptera: Liposcelididae)

Two nicotinic acetylcholine receptor (nAChR) subunit genes, Lbα1 and Lbα8, were isolated and characterized from psocid, Liposcelis bostrychophila Badonnel, using the rapid amplification of cDNA ends (RACE) technique. They are the first two nAChR family members isolated from the insect order of Psocoptera. The full‐length cDNAs of Lbα1 (GenBank accession number: EU871527) and Lbα8 (EU871526) consist of 2,025 and 1,763 nucleotides, respectively, and an open reading frame of 1,644 and 1,608 bp encoding 547 and 535 amino acid proteins, respectively. Both genes have typical features of nAChR family members, though they share only 56% identity in amino acid sequence. The dendrogram generated by the MEGA 3.1 program shows that the protein deduced by Lbα1 had the closest phylogenetic relationship to Agamα1 from Anopheles gambiae and Amelα1 from Apis mellifera, and Lbα8 shares the highest identity with Agamα8 from An. gambiae and Amelα8 from A. mellifera. Quantitative real‐time PCR analysis showed that Lbα1 was expressed 2.03–6.54‐fold higher than Lbα8 at the different developmental stages of L. bostrychophila. The highest expression levels of Lbα1 and Lbα8 were both detected at adult stage and the lowest were at the third and fourth nymphal stages, respectively. There was a stable and relatively low expression level for Lbα1, whereas there was a descending expression pattern for Lbα8 in the 1st through the 4th nymphal stadia. © 2009 Wiley Periodicals Inc.     

First identification of a chemotactic receptor in an invertebrate species: structural and functional characterization of Ciona intestinalis C3a receptor

In mammals, the bioactive fragment C3a, released from C3 during complement activation, is a potent mediator of inflammatory reactions and exerts its functional activity through the specific binding to cell surface G protein-coupled seven-transmembrane receptors. Recently, we demonstrated a Ciona intestinalis C3a (CiC3a)-mediated chemotaxis of hemocytes in the deuterostome invertebrate Ciona intestinalis and suggested an important role for this molecule in inflammatory processes. In the present work, we have cloned and characterized the receptor molecule involved in the CiC3a-mediated chemotaxis and studied its expression profile. The sequence, encoding a 95,394 Da seven-transmembrane domain protein, shows the highest sequence homology with mammalian C3aRs. Northern blot analysis revealed that the CiC3aR is expressed abundantly in the heart and neural complex and to a lesser extent in the ovaries, hemocytes, and larvae. Three polyclonal Abs raised in rabbits against peptides corresponding to CiC3aR regions of the first and second extracellular loop and of the third intracellular loop react specifically in Western blotting with a single band of 98-102 kDa in hemocyte protein extracts. Immunostaining performed on circulating hemocytes with the three specific Abs revealed that CiC3aR is constitutively expressed only in hyaline and granular amoebocytes. In chemotaxis experiments, the Abs against the first and second extracellular loop inhibited directional migration of hemocytes toward the synthetic peptide reproducing the CiC3a C-terminal sequence, thus providing the compelling evidence that C. intestinalis expresses a functional C3aR homologous to the mammalian receptor. These findings further elucidate the evolutionary origin of the vertebrate complement-mediated proinflammatory process.     

Genome-wide identification of neuropeptides and their receptor genes in Bemisia tabaci and their transcript accumulation change in response to temperature stresses

Insect neuropeptides play an important role in regulating physiological functions such as growth,development,behavior and reproduction.We identified temperaturesensitive neuropeptides and receptor genes of the cotton whitefly,Bemisia tabaci.We identified 38 neuropeptide precursor genes and 35 neuropeptide receptors and constructed a phylogenetic tree using additional data from other insects.As temperature adaptability enables B.tabaci to colonize a diversity of habitats,we performed quantitative polymerase chain reaction with two temperature stresses(low=4℃ and high=40℃)to screen for temperature-sensitive neuropeptides.We found many neuropeptides and receptors that may be involved in the temperature adaptability of B.tabaci.This study is the first to identify B.tabaci neuropeptides and their receptors,and it will help to reveal the roles of neuropeptides in temperature adaptation of B.tabaci.     

Molecular characterization of calcitonin gene-related peptide (CGRP) in a rat medullary thyroid carcinoma cell line

The rat medullary thyroid carcinoma cell line, CA-77, is known to express the calcitonin gene and the cell line has been used for characterization of procalcitonin. The present investigations concentrate on a molecular characterization of the calcitonin gene-related peptide (CGRP) expressed by a subclone of this cell line. The investigations demonstrate that this subclone produces significantly more CGRP compared to calcitonin. Gel chromatography of cell extracts demonstrates heterogeneity for both CGRP and calcitonin, but a significant amount of immunoreactivity elutes corresponding to the elution position for synthetic CGRP and calcitonin, respectively. The gel chromatogram for CGRP demonstrates four immunoreactive peaks with K_d of 0.42, 0.53, 0.68, and 0.85. The immunoreactive peak with K_d 0.42 elutes corresponding to synthetic rat CGRP. The four immunoreactive peaks were characterized by high pressure liquid chromatography followed by sequence analysis and mass spectrometry. The immunoreactive peak with K_d 0.42 was identified as rat -CGRP as was the peak with K_d 0.53. The peak with K_d 0.68 was identified as 19–37 rat -CGRP and the peak with K_d 0.85 as 28–37 rat -CGRP. In summary, we find that the CA-77 cell line expresses large quantities of normally processed amidated -CGRP and specific fragments thereof. However, the cell line does not express detectable levels of rat β-CGRP. The findings indicate that the CA-77 cell line can be useful for studies of calcitonin/CGRP gene expression.     

Molecular cloning, characterization and expression analysis of an IL-21 homologue from Tetraodon nigroviridis

Interleukin-21 (IL-21) is an important immune cytokine that was well characterized in human and mammals, but little is known in fish. In present study, an IL-21 homologue was cloned and well characterized from Tetraodon nigroviridis. The full-length Tetraodon IL-21 cDNA was 849bp in size, containing an open reading frame (ORF) of 438bp that translated a 145 amino-acid peptide, a 5' untranslated region (UTR) of 69bp, and a 3' UTR of 342bp. The deduced peptide shared identity of 20-49% with other known IL-21 sequences. The Tetraodon IL-21 gene had six exons while both human and Takifugu IL-21 gene contained only five exons. However, the level of synteny between human, Takifugu and Tetraodon genomes was well conserved during evolution. In vivo expression study showed that Tetraodon IL-21 mRNAs were constitutively expressed at a low level and only in limited tissues, including gut, gill and gonad in healthy fish, and stimulation with LPS increased the expression of IL-21 in these tissues and induced the expression of IL-21 in kidney, spleen and skin, indicating that IL-21 is an inflammatory stress inducible gene associated with the anti-bacterial defense in fish. Our study provided further evidence for the existence of IL-21 in fish, and gained further insight into the immunological functions of IL-21 gene in fish.     

Pharmacological characterization of a serotonin receptor involved in an early embryonic behavior of Helisoma trivolvis

In contrast to the abundance of information on the many physiological and developmental actions of serotonin in molluscan nervous systems, comparatively little is known about the serotonin receptors involved in these responses. Embryos of the pulmonate gastropod, Helisoma trivolvis, display a cilia-driven rotational behavior that is regulated by endogenous serotonin. In the present study, two functional assays were used to determine some of the pharmacological properties of the receptors that mediate the cilio-excitatory action of serotonin. Timelaspe video microscopy was used to measure whole embryo rotation rat and cilia beat frequency in isolated cells. In dose-response experiments, serotonin was approximately 10 times more potent in stimulating cilia beat frequency over embryo rotation. In rotation experiments, 5-carboxyamidotryptamine and methysergide had effective agonist activity in dose ranges similar to that of serotonin (1 to 100 μM). In contrast, 8-hydroxydiproylaminotetralin HBr (8-OH-DPAT) displayed agonist activity of lower potency and effectiveness. Several compounds displayed antagonist activity in the 1 to 100 μM dose range, including mianserin, spiperone, ritanserin, 1-(1-naphthyl) piperazine, and Propranolol. α-Methylserotonin had mixed agonist–antagonist activity, and metoclopramide, MDL-72222, and ketanserin were inactive. Experiments on isolated cells suggested that the extremely effective antagonism displayed by mianserin in the embryo rotation assay was due to its specific activity at ciliary serotonin receptors. These results implicate the presence of a novel serotonin receptor on embryonic ciliated cells that is pharmacologically distinct from those previously characterized in vertebrate or invertebrate systems. 1994 John Wiley & Sons, Inc.     

BDV核蛋白FQ RT-PCR试剂盒的评价与应用

为评价博尔纳病病毒(Borna disease virus,BDV)核蛋白荧光定量PCR(FQRT-PCR)试剂盒的各项指标,比较分子信标探针相对普通探针的优势,并了解其实际检测效果,本课题组使用BDVOL持续感染细胞株、非BDV病毒序列转染的OL细胞、正常的OL细胞,对BDVRT-PCR试剂盒的敏感性、特异性、重复性和稳定性进行评估,同时检测部分临床病人和动物外周血液RNA。实验结果显示:试剂盒可以检测出的病毒RNA最低浓度为2.5×101,相当于1个病毒拷贝数,无非特异检出;不同批次的试剂盒的检测结果变异系数小于0.7;加速破坏的试剂盒和正常试剂盒检测结果之间变异系数在2以内;对临床病人检测阳性率为3.6%,对动物检测阳性率为4.2%(猪)和1.5%(马)。可见该试剂盒重复性和稳定性均好;敏感性、特异性优于普通探针试剂盒,是BDV基础研究、流行病学调查和临床检测的良好工具。     

Molecular and phylogenetic characterization of potentially toxic cyanobacteria in Tunisian freshwaters

This study presents a genetic characterization of 27 potentially toxic cyanobacterial strains isolated from seven reservoirs located in the north and centre of Tunisia. These strains belonged mainly to Microcystis aeruginosa, Cylindrospermopsis raciborskii and Planktothrix agardhii species. Their toxicological potential was evaluated by molecular biology tools, which showed that none of the isolated strains carried segments of the gene cluster responsible for the production of cylindrospermopsin and saxitoxin. The majority of Microcystis isolates were able to synthesize microcystin, since they presented the six characteristic segments of the microcystin synthetase mcy cluster (mcyA, -B, -C, -D, -E and -G). This was further confirmed by MALDI-TOF analysis that showed the presence of eight microcystin variants, including microcystin-LR. The taxonomic identification of the strains was assessed based on the variability of the 16S rRNA gene sequences. Furthermore, the 16S-23S rRNA ITS sequences of Microcystis isolates and rpoC1 sequences of Cylindrospermopsis strains were also used in the phylogenetic analysis.     

Molecular characterization of CCR6: Involvement of multiple domains in ligand binding and receptor signaling

The CC chemokine receptor 6 (CCR6) is selectively expressed on memory T cells, B cells, and dendritic cells and appears to be involved in the initiation of a memory immune response. The only chemokine ligand for CCR6 is CCL20/MIP-3. In the present study, we attempted to define the extracellular domains (ECDs) of CCR6 responsible for CCL20/MIP-3 binding using a domain-swapping approach in which the ECDs of CCR6 were substituted with the corresponding CCR5 domains to generate various CCR6/CCR5 chimeras. These chimeras were tested for receptor expression, ligand binding, and functional activity as evaluated by calcium flux and chemotaxis. All chimeras showed respectable surface expression; however only one, substituted with extracellular loop 1 from CCR5, showed reduced functional activity. The general failure of functionality of the CCR6/CCR5 chimeras may imply that characteristics of each ECD are critical for coordination among all the ECDs of CCR6. Additionally, of interest, a chimera containing all of the ECDs from CCR5 in the context of CCR6 neither responded to CCR5 ligands nor served as a coreceptor for macrophage-tropic HIV-1. These results suggest that not only ECDs but also transmembrane and intracellular domains of CCR5 are involved in both ligand binding and coreceptor activity.     

Molecular assessment on impact of uranium ore contamination in soil bacterial diversity

Impact of uranium (U) ore and soluble uranium (at pH 4.0) contamination on agricultural soil bacterial diversity was assessed by using laboratory microcosms for one year. Diversity and abundance of metabolically active bacterial populations in periodically collected microcosm’s samples were analyzed by extracting total RNA and preparation of cDNA followed by analysis of 16S rRNA gene by DGGE and real time PCR. DGGE analysis revealed prominent shift of soil bacterial population due to uranium ore contamination within 12 months while uranium ore along with soluble U completely destroyed the soil bacterial diversity within first six months. Real time PCR based analysis indicated 100–200 folds increase in 16S rRNA gene copies of total as well as individual bacterial taxa in both U ore amended and unamended soils in first six months while increase in incubation period upto 12 months showed reduction of the same only in U ore amended soil. Antagonistic effect of U ore contamination on soil bacterial diversity indicated the severe impact of U mining likely to have on nearby ecosystems. Role of U at acidic pH in destroying the diversity completely is noteworthy as it corroborated the disastrous consequence of acid mine drainage generated from U mine sites.     

Molecular characterization of an Arabidopsis thaliana cDNA coding for a monofunctional aspartate kinase

A cDNA clone encoding a monofunctional aspartate kinase (AK, ATP:L-aspartate 4-phosphotransferase, EC 2.7.2.4) has been isolated from an Arabidopsis thaliana cell suspension cDNA library using a homologous PCR fragment as hybridizing probe. Amplification of the PCR fragment was done using a degenerate primer designed from a conserved region between bacterial monofunctional AK sequences and a primer identical to a region of the A. thaliana bifunctional aspartate kinase-homoserine dehydrogenase (AK-HSDH). By comparing the deduced amino acid sequence of the fragment with the bacterial and yeast corresponding gene products, the highest identity score was found with the Escherichia coli AKIII enzyme that is feedback-inhibited by lysine (encoded by lysC). The absence of HSDH-encoding sequence at the COOH end of the peptide further implies that this new cDNA is a plant lysC homologue. The presence of two homologous genes in A. thaliana is supported by PCR product sequences, Southern blot analysis and by the independent cloning of the corresponding second cDNA (see Tang et al., Plant Molecular Biology 34, pp. 287–294 [this issue]). This work is the first report of cloning a plant putative lysine-sensitive monofunctional AK cDNA. The presence of at least two genes is discussed in relation to possible different physiological roles of their respective product.