Nitric oxide: a co-modulator of efferent peptidergic neurosecretory cells including a unique octopaminergic neurone innervating locust heart

Our findings suggest that nitric oxide (NO) acts as peripheral neuromodulator in locusts, in which it is commonly co-localized with RF-like peptide in neurosecretory cells. We also present the first evidence for NO as a cardio-regulator in insects. Putative NO-producing neurones were detected in locust pre-genital free abdominal ganglia by NADPH-diaphorase histochemistry and with an antibody against NO synthase (NOS). With both methods, we identified the same 14 somata in each examined ganglion: two dorsal posterior midline somata; six ventral posterior midline somata; and three pairs of lateral somata. A combination of NOS-detection methods with nerve tracing and transmitter immunocytochemistry revealed that at least 12 of these cells were efferent, of which four were identified as peptidergic neurosecretory cells with an antiserum detecting RFamide-like peptides. One of the latter was unequivocally identified as an octopaminergic dorsal unpaired median (DUM) neurone, which specifically projected to the heart (“DUM-heart”). Its peripheral projections revealed by axon tracing appeared as a meshwork of varicose endings encapsulating the heart. NOS-like immunoreactive profiles were found in the heart nerve. NO donors caused a dose-dependent increase in heart rate. This cardio-excitatory effect was negatively correlated to resting heart rate and seemed to be dependent on the physiological state of the animal. Hence, NO released from neurones such as the rhythmically active DUM-heart might exert continuous control over the heart. Possible mechanisms for the actions of NO on the heart and interactions with other neuromodulators co-localized in the DUM-heart neurone (octopamine, taurine, RF-amide-like peptide) are discussed.P.A.S. is the leading author, following the tragic death of Alexander Bullerjahn on 20.12.03.This work was supported by the Deutsche Forschungsgemeinschaft (grant number SFB 515; project of H.-J.P.).     

Gene expression and immunolocalisation of a calcium-activated chloride channel during the stratification of cultivated and developing corneal epithelium

The spatial and temporal localisation of a calcium-activated chloride channel (CLCA) and its mRNA was investigated, during the in vivo and in vitro development of stratified epithelia, by fluorescence immunohistochemistry and quantitative polymerase chain reaction in embryonic chicken corneas and the expansion of excised human corneal stem cells on amniotic membrane. Single-layered human epithelial cultures on amniotic membrane and early day embryonic chicken corneas expressed relatively little human CLCA2 or its chicken homologue. However, as the epithelium in both models matured and the number of cell-layers increased, the gene expression level and protein staining intensity increased, primarily within the basal cells of both the cultured and embryonic tissues. These results demonstrate that human CLCA2 protein and mRNA expression are elevated during epithelial stratification, suggesting that this protein plays a role in the growth of multi-layered corneal epithelia during both natural development and tissue cultivation. This work was supported by the Japanese Society for the Promotion of Science (CJC) and The Royal Society (C.J.C.).     

Insecticidal and antifeedant activities of proteins secreted by entomopathogenic fungi against Spodoptera littoralis (Lep., Noctuidae)

Abstract:  Entomopathogenic fungi are a poorly exploited source of insecticidal proteins, which may be used for the development of new natural insecticides and as alternative molecules for transgenic deployment. The crude soluble protein extracts in Adamek's liquid medium of 25 fungal isolates belonging to the fungal species Metarhizum anisopliae , Beauveria bassiana , Beauveria brongniartii and Scopulariopsis brevicaulis were screened by per os toxicity on Spodoptera littoralis larvae. Whilst extracts from two M. anisopliae and two B. bassiana isolates gave significant mortalities when applied either on alfalfa leaf discs or incorporated into artificial diet, the one from M. anisopliae 01/58-Su isolate was the only most toxic that showed promise for S. littoralis control. In leaf disc assays, this extract exhibited strong dose-related toxic and antifeedant activity against the larvae. At 10, 20 and 40  μ g protein/insect, the extract gave 61.3%, 96.6% and 100.0% mortality, respectively, and average survival time of 5.7, 4.3 and 3.1 days respectively. Not only the antifeeding index was dose-related, but it significantly increased over time in a dose-related manner. Longer exposure times led to a dose-related significant increase in larval mortality. The exposure times for 50% mortality were 91.3 h and 62.1 h for 20 and 40  μ g protein/insect respectively. The crude extract when exposed to higher temperature or protease treatment lost toxicity, indicating that toxicity was protein mediated. In addition, the liquid medium composition did not influence its insecticidal activity. The effects of the protein extract on midgut cells of second instar larvae of S. littoralis were investigated by using both light and electron microscopy. A progressive bleeding of the midgut epithelium into the gut lumen was observed along with lysis of the epithelium and deterioration of the microvilli.     

16S rDNA用作荧光定量PCR靶基因快速检测铜绿假单胞菌

对20余种细菌16SrDNAs进行多序列比对与进化树分析,设计铜绿假单胞菌(Pseudomonasaeruginosa,PA)荧光定量PCR(fluorescencequantitativePCR,FQ-PCR)特异性引物。提取PA基因组DNA,以特异性引物扩增16SrDNA靶片段,并构建重组质粒pMDT-Pfr。将梯度稀释的pMDT-Pfr质粒作为模板,用于建立定量标准曲线。以SYBRGreenI荧光染料建立20μL反应体系,对不同浓度的PADNA样品进行FQ-PCR检测。同时,以金黄色葡萄球菌、伤寒杆菌、福氏志贺菌、变形杆菌、表皮葡萄球菌、大肠杆菌和结核杆菌的基因组DNA作阴性对照,验证FQ-PCR方法检测PA的特异性。结果显示,设计的FQ-PCR引物的靶向序列,仅对PA16SrDNA有高度同源性;FQ-PCR方法检测PA,其灵敏度达3.6pg/μL的基因组DNA或(2.1×103±3.1×102)拷贝/μL的16SrDNA基因,并且具有很强的特异性;从细菌DNA提取到FQ-PCR检测,可在2h左右完成PA鉴定。较传统的培养鉴定法而言,以16SrDNA作为FQ-PCR靶基因快速检测PA,具有很好的研究价值与应用前景。     

Real-time PCR for quantification of aerobic anoxygenic phototrophic bacteria based on pufM gene in marine environment

The abundance of aerobic anoxygenic phototrophic bacteria (AAPB), a new functional group that plays important roles in marine carbon cycling, is determined frequently by infrared epifluorescence microscopic analysis (IREM) or high-performance liquid chromatography (HPLC) based on detecting BChl a (bacteriochlorophyll a) fluorescence signal at 880 nm. Unfortunately, the fluorescence signal is often influenced by environmental variables and physiological state of cell. Here we developed a real-time quantitative PCR (qPCR) assay based on pufM gene to specifically quantify AAPB in marine environments. High specificity and sensitivity for estimation of AAPB abundance were revealed by analysis of amplification products, melting curves and target sequences. The phylogenetic tree indicated that this primer set is suitable for a wide genetic diversity of AAPB, including α-3, α-4 Proteobacteria and clones of unclear taxonomic position. In contrast, no amplicon was obtained from green non-sulphur bacteria and oxygenic phototrophic bacteria such as Cyanobacterial genomic DNA. The melting behavior could indicate predominant phenotypes in AAPB community in addition to validating the products of qPCR. The AAPB was estimated to range from 1.3 × 10⁴ cell/ml to 3.4 × 10⁵ cell/ml in our 10 tested water samples by this qPCR assay. Further investigations on the abundance distribution of AAPB in marine environments using the qPCR assay may provide new insight into their ecological functions.     

Mutants and isolates of Metarhizium anisopliae are diverse in their relationships between conidial pigmentation and stress tolerance

Conidial pigmentation is involved in protection against heat and UV radiation in several fungal species. In this study, we compare the tolerance of 17 color mutants of wild-type ARSEF 23 plus 13 color mutants of wild-type ARSEF 2575 of Metarhizium anisopliae var. anisopliae to wet-heat and UV-B or simulated-solar radiation. The stress tolerance of each mutant was compared with that of its wild-type parent, and with the most thermo- and UV-tolerant wild-type Metarhizium we have tested to date, M. anisopliae var. acridum (ARSEF 324). The color of each isolate or mutant was identified with the PANTONE Color Standard book [Eiseman, L., Herbert, L., 1990. The PANTONE((R)) Book of Color: over 1000 color standards: color basics and guidelines for design, fashion, furnishing... and more. Harry N. Abrams, Inc., Publishers, New York]. In addition, the pigments of each mutant or wild-type were extracted and the UV absorbances of the extracts compared to the stress tolerance of those isolates; but no relationships were detected. Color mutants of ARSEF 23, in general, were less UV tolerant than their parent wild-type. With ARSEF 23 and its mutants, conidial pigmentation was important to conidial tolerance to UV-B and simulated-solar radiation; but color had less impact on ARSEF 2575 and its mutants. The ARSEF 2575 color mutants were less variable in UV tolerance than those of ARSEF 23, even though very similar colors occurred in the two groups of mutants. When color mutants of ARSEF 23 reverted to wild-type color they recovered wild-type levels of UV tolerance. Results of UV-B and UV-A exposures of wild-types ARSEF 23 and ARSEF 2575 conidia indicated that they are equally tolerant of UV-A, but differ in UV-B-response. For thermotolerance, several mutants were more heat tolerant than their wild-type parents. Accordingly, darker pigmentation of wild-type isolates was not important to protection against heat.     

Cell cycle-dependent regulation of kainate-induced inward currents in microglia

Microglia are reported to have α-amino-hydroxy-5-methyl-isoxazole-4-propionate/kainate (KA) types. However, only small population of primary cultured rat microglia (approximately 20%) responded to KA. In the present study, we have attempted to elucidate the regulatory mechanism of responsiveness to KA in GMIR1 rat microglial cell line. When the GMIR1 cells were plated at a low density in the presence of granulocyte macrophage colony-stimulating factor, the proliferation rate increased and reached the peak after 2 days in culture and then gradually decreased because of density-dependent inhibition. At cell proliferation stage, approximately 80% of the GMIR1 cells exhibited glutamate (Glu)- and KA-induced inward currents at cell proliferation stage, whereas only 22.5% of the cells showed responsiveness to Glu and KA at cell quiescent stage. Furthermore, the mean amplitudes of inward currents induced by Glu and KA at cell proliferation stage (13.8 ± 3.0 and 8.4 ± 0.6 pA) were significantly larger than those obtained at cell quiescent stage (4.7 ± 0.8 and 6.2 ± 1.2 pA). In the GMIR1 cells, KA-induced inward currents were markedly inhibited by (RS)-3-(2-carboxybenzyl) willardiine (UBP296), a selective antagonist for KA receptors. The KA-responsive cells also responded to (RS)-2-amino-3-(3-hydroxy-5-tert-butylisoxazol-4-yl) propanoic acid (ATPA), a selective agonist for GluR5, in both GMIR1 cells and primary cultured rat microglia. Furthermore, mRNA levels of the KA receptor subunits, GluR5 and GluR6, at the cell proliferation stage were significantly higher than those at the cell quiescent stage. Furthermore, the immunoreactivity for GluR6/7 was found to increase in activated microglia in the post-ischemic hippocampus. These results strongly suggest that microglia have functional KA receptors mainly consisting of GluR5 and GluR6, and the expression levels of these subunits are closely regulated by the cell cycle mechanism.