New functions of histamine found in histidine decarboxylase gene knockout mice

Gene targeting techniques have revolutionized the investigation of the effects of bioactive substances in pathological and physiological conditions. Histamine synthesis is uniquely catalyzed by L-histidine decarboxylase. The knockout mice of this gene express no histamine-producing activity and lack histamine. These mice have been used to examine the mechanisms of histamine in several known phenotypes, e.g., gastric acid secretion, contraction of smooth muscles, vascular permeability, and awakening, and have also been used to explore unreported effects of histamine in the whole body. First, we will review the former mechanisms and then move to the latter, new effects. Especially, in the latter mechanisms, we focus on several important roles of histamine in angiogenesis, neutrophil and eosinophil recruitment, bacterial infection, and systemic anaphylaxis in this review. Moreover, to our surprise, the morphology of mast cells in the knockout mice was severely affected by the absence of histamine in terms of their granules.     

Locations of crossover breakpoints within the CMT1A-REP repeat in Japanese patients with CMT1A and HNPP

We have used human β2 and β4 cDNA probes to map the genes encoding two isoforms of the regulatory β subunit of voltage-activated Ca²⁺ channels, viz. CACNB2 (β2) and CACNB4 (β4), to human chromosomes 10p12 and 2q22-q23, respectively, by fluorescence in situ hybridization. The gene encoding the β2 protein, first described as a Lambert-Eaton myasthenic syndrome (LEMS) antigen in humans, is found close to a region that undergoes chromosome rearrangements in small cell lung cancer, which occurs in association with LEMS. CACNB2 (β2) and CACNB4 (β4) genes are members of the ion-channel gene superfamily and it should now be possible to examine their loci by linkage analysis of ion-channel-related disorders. To date, no such disease-related gene has been assigned to 10p12 and 2q22-q23. Received: 5 February 1997 / Accepted: 4 April 1997     

Genetic tools for select-agent-compliant manipulation of Burkholderia pseudomallei

Because of Burkholderia pseudomallei's classification as a select agent in the United States, genetic manipulation of this bacterium is strictly regulated. Only a few antibiotic selection markers, including gentamicin, kanamycin, and zeocin, are currently approved for use with this bacterium, but wild-type strains are highly resistant to these antibiotics. To facilitate routine genetic manipulations of wild-type strains, several new tools were developed. A temperature-sensitive pRO1600 broad-host-range replicon was isolated and used to construct curable plasmids where the Flp and Cre recombinase genes are expressed from the rhamnose-regulated Escherichia coli P(BAD) promoter and kanamycin (nptI) and zeocin (ble) selection markers from the constitutive Burkholderia thailandensis ribosomal P(S12) or synthetic bacterial P(EM7) promoter. Flp and Cre site-specific recombination systems allow in vivo excision and recycling of nptII and ble selection markers contained on FRT or loxP cassettes. Finally, expression of Tn7 site-specific transposase from the constitutive P1 integron promoter allowed development of an efficient site-specific chromosomal integration system for B. pseudomallei. In conjunction with a natural transformation method, the utility of these new tools was demonstrated by isolating an unmarked delta(amrRAB-oprA) efflux pump mutant. Exploiting natural transformation, chromosomal DNA fragments carrying this mutation marked with zeocin resistance were transferred between the genomes of two different B. pseudomallei strains. Lastly, the deletion mutation was complemented by a chromosomally integrated mini-Tn7 element carrying the amrAB-oprA operon. The new tools allow routine select-agent-compliant genetic manipulations of B. pseudomallei and other Burkholderia species.     

IL-27 affects helper T cell responses via regulation of PGE2 production by macrophages

IL-27 is a heterodimeric cytokine that regulates both innate and adaptive immunity. The immunosuppressive effect of IL-27 largely depends on induction of IL-10-producing Tr1 cells. To date, however, effects of IL-27 on regulation of immune responses via mediators other than cytokines remain poorly understood. To address this issue, we examined immunoregulatory effects of conditional medium of bone marrow-derived macrophages (BMDMs) from WSX-1 (IL-27Rα)-deficient mice and found enhanced IFN-γ and IL-17A secretion by CD4⁺ T cells as compared with that of control BMDMs. We then found that PGE₂ production and COX-2 expression by BMDMs from WSX-1-deficient mice was increased compared to control macrophages in response to LPS. The enhanced production of IFN-γ and IL-17A was abolished by EP2 and EP4 antagonists, demonstrating PGE₂ was responsible for enhanced cytokine production. Murine WSX-1-expressing Raw264.7 cells (mWSX-1-Raw264.7) showed phosphorylation of both STAT1 and STAT3 in response to IL-27 and produced less amounts of PGE₂ and COX-2 compared to parental RAW264.7 cells. STAT1 knockdown in parental RAW264.7 cells and STAT1-deficiency in BMDMs showed higher COX-2 expression than their respective control cells. Collectively, our result indicated that IL-27/WSX-1 regulated PGE₂ secretion via STAT1–COX-2 pathway in macrophages and affected helper T cell response in a PGE₂-mediated fashion.     

Stereoselective synthesis of (22R)- and (22S)-castasterone/ponasterone A hybrid compounds and evaluation of their molting hormone activity

Two stereoisomers of a castasterone/ponasterone A hybrid compound, the (20R,22R) and (20R,22S)-isomers of 2alpha,3alpha,20,22-tetrahydroxy-5alpha-cholestan-6-one, were synthesized stereoselectively and their binding activity to the ecdysteroid receptor was determined. From the concentration-response curve for the inhibition of the incorporation of tritiated ponasterone A into ecdysteroid receptor containing insect cells, the concentration (IC50) required to inhibit 50% of the incorporation of radioactivity into cells was evaluated. The IC50 values of the (22R)- and (22S)-isomers were determined to be 0.30 and 38.9 microM against Kc cells, respectively, indicating that the (22R)-isomer is about 100 times more potent than the corresponding (22S)-isomer. IC50 values of these compounds against lepidopteran Sf-9 cells were determined to be 0.36 and 12.9 microM, respectively. The molting hormonal effect was examined in a Chilo suppressalis integument system and the 50% effective concentration for the stimulation of N-acetylglucosamine incorporation into the cultured integument was determined to be 2.7 microM for the (22R)-isomer, while the (22S)-isomer was inactive. On the other hand, both isomers did not show brassinolide-like activity in the rice lamina inclination assay.     

Identification of a second DNA binding site in the human Rad52 protein

Rad52 plays essential roles in homology-dependent double-strand break repair. Various studies have established the functions of Rad52 in Rad51-dependent and Rad51-independent repair processes. However, the precise molecular mechanisms of Rad52 in these processes remain unknown. In the present study we have identified a novel DNA binding site within Rad52 by a structure-based alanine scan mutagenesis. This site is closely aligned with the putative single-stranded DNA binding site determined previously. Mutations in this site impaired the ability of the Rad52-single-stranded DNA complex to form a ternary complex with double-stranded DNA and subsequently catalyze the formation of D-loops. We found that Rad52 introduces positive supercoils into double-stranded DNA and that the second DNA binding site is essential for this activity. Our findings suggest that Rad52 aligns two recombining DNA molecules within the first and second DNA binding sites to stimulate the homology search and strand invasion processes.     

Characteristics of rabbit ClC-2 current expressed in Xenopus oocytes and its contribution to volume regulation

In theXenopus oocyte heterologous expressionsystem, the electrophysiological characteristics of rabbit ClC-2current and its contribution to volume regulation were examined.Expressed currents on oocytes were recorded with a two-electrodevoltage-clamp technique. Oocyte volume was assessed by taking picturesof oocytes with a magnification of ×40. Rabbit ClC-2 currentsexhibited inward rectification and had a halide anion permeabilitysequence of Cl  Br  I  F. ClC-2 currents wereinhibited by 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB),diphenylamine-2-carboxylic acid (DPC), and anthracene-9-carboxylic acid(9-AC), with a potency order of NPPB > DPC = 9-AC, but were resistant to stilbene disulfonates. These characteristics are similarto those of rat ClC-2, suggesting rabbit ClC-2 as a counterpart of ratClC-2. During a 30-min perfusion with hyposmolar solution, currentamplitude at 160 mV and oocyte diameter were compared amongthree groups: oocytes injected with distilled water, oocytes injectedwith ClC-2 cRNA, and oocytes injected with ClC-2NT cRNA (an openchannel mutant with NH₂-terminaltruncation). Maximum inward current was largest in ClC-2NT-injectedoocytes (5.9 ± 0.4 µA), followed by ClC-2-injected oocytes(4.3 ± 0.6 µA), and smallest in water-injected oocytes(0.2 ± 0.2 µA), whereas the order of increase in oocytediameter was as follows: water-injected oocytes (9.0 ± 0.2%) > ClC-2-injected oocytes (5.3 ± 0.5%) > ClC-2NT-injected oocytes (1.1 ± 0.2%). The findings that oocyte swelling wassmallest in oocytes with the largest expressed currents suggest thatClC-2 currents expressed in Xenopusoocytes appear to act for volume regulation when exposed to ahyposmolar environment.

     

Cleome hassleriana plants fully support the development and reproduction of Nesidiocoris tenuis

BioControl - Nesidiocoris tenuis (Reuter) (Hemiptera: Miridae) is a zoophytophagous predator that feeds on plants as well as prey. Several non-crop host plant species have been used to maintain...