Increased in vitro responses of tracheal smooth muscle from hyperresponsive guinea pigs

Repeated aerosol antigen challenge of previously sensitized guinea pigs induces airway hyperresponsiveness to inhaled acetylcholine. To determine the mechanism producing these airway changes and assuming that changes in the trachealis muscle reflect changes in muscle of the entire tracheobronchial tree, we examined the in vitro smooth muscle mechanics and morphometric parameters of tracheae from guinea pigs demonstrating hyperresponsiveness in vivo vs. tracheae from control guinea pigs. No differences between these groups were found in luminal volume at zero transmural pressure, passive pressure-volume characteristics, or area of airway wall. Smooth muscle areas were slightly less in tracheae from hyperresponsive guinea pigs. Tracheae from hyperresponsive guinea pigs had both significantly increased isovolumetric force generation and isobaric shortening compared with tracheae from controls when evaluated over the range of transmural pressures from -40 to 40 cmH2O. We conclude that the in vivo airway hyperresponsiveness induced with repeated antigen challenge is associated with both increased force generation and shortening of tracheal smooth muscle without increased muscle mass, suggesting enhanced contractile activity.     

Detection and Identification of Yersinia pestis by Polymerase Chain Reaction (PCR) Using Multiplex Primers

A PCR method for detection of Yersinia pestis-virulence determinants by the use of multiplex primers was developed. Four pairs of oligonucleotide primers were designed from each gene of three kinds of virulent plasmids and a chromosomal DNA; 60-Md plasmid-located gene (caf1) encoding Y. pestis-specific capsular antigen fraction 1, a Y. pestis-specific region of a yopM gene encoded on 42-Md virulent plasmid, a plasminogen activator gene (pla) encoded on Y. pestis-specific 7-Md plasmid and an invasin protein gene (inv) encoded on chromosomal DNA. This multiplex-primer system was specific for the detection of Y. pestis among pathogenic Yersinia species and other enterobacteriaceae having antigens common to Y. pestis. Since this method is simple and safe, it will be useful to identify and confirm Y. pestis in cases of emergency and for the surveillance of epidemics.     

Indolepyruvate Pathway for Indole-3-Acetic Acid Biosynthesis in Bradyrhizobium elkanii

Indole-3-acetaldehyde (IAAId) was detected in the culture supernatantof Bradyrhizobium elkanii. Deuteriumlabelled L-tryptophan (Trp)was incorporated into IAAId and indole-3-acetic acid (IAA),suggesting that B. elkanii produces IAA via IAAId from Trp.In B. elkanii cell suspension, indole-3-pyruvic acid (IPyA)was converted to IAAId, and exogenously added IAAId was rapidlyconverted to IAA. Furthermore, the activity of indolepyruvatedecarboxylase (IPDC), which catalyzes the decarboxylation ofIPyA to produce IAAId and is a key enzyme for IPyA pathway,was detected in B. elkanii cell-free extract. The IPDC activitydepended on Mg²⁺ and thiamine pyrophosphate, cofactors of decarboxylation.This mounting evidence strongly suggests that IAA synthesisoccurs via IPyA pathway (Trp IPyA p IAAId IAA) in B. elkanii. (Received December 11, 1995; Accepted March 4, 1996)     

Characterization of cis-regulatory elements of the c-myc promoter responding to human GM-CSF or mouse interleukin 3 in mouse proB cell line BA/F3 cells expressing the human GM-CSF receptor.

Interleukin 3 (IL-3) or granulocyte macrophage colony-stimulating factor (GM-CSF) activates c-fos, c-jun, and c-myc genes and proliferation in both hematopoietic and nonhematopoietic cells. Using a series of deletion mutants of the beta subunit of human GM-CSF receptor (hGMR) and inhibitors of tyrosine kinase, two distinct signaling pathways, one for activation of c-fos and c-jun genes, and the other for cell proliferation and activation of c-myc gene have been elucidated. In contrast to wealth of information on the pathway leading to activation of c-fos/c-jun genes, knowledge of the latter is scanty. To clarify the mechanisms of activation of c-myc gene by cytokines, we established a transient transfection assay in mouse proB cell line BA/F3 cells expressing hGMR. Analyses of hGMR beta subunit mutants revealed two cytoplasmic regions involved in activation of the c-myc promoter, one is essential and the other is dispensable but enhances the activity. These regions are located at the membrane proximal and the distal regions covering amino acid positions 455-544 and 544-589, respectively. Characterization of cis-acting regulatory elements of the c-myc gene showed that the region containing the P2 promoter initiation site is sufficient to mediate the response to mIL-3 or hGM-CSF. Electrophoretic mobility shift assay using an oligonucleotide corresponding to the distal putative E2F binding site revealed that p107/E2F complex, the negative regulator of E2F, decreased, and free E2F increased after mIL-3 stimulation. These results support the thesis that mIL-3 or hGM-CSF regulates the c-myc promoter by altering composition of the E2F complexes at E2F binding site.     

Cloning and sequence analysis of theMaackia amurensis haemagglutinin cDNA

Maackia amurensis haemagglutinin (MAH) is a leguminous lectin which preferentially binds to a cluster of sialylatedO-linked carbohydrate chains (Konami Y, Yamamoto K, Osawa T, Irimura T (1994)FEBS Lett 342:334–38). In the present study a 950 bp cDNA clone encoding MAH was isolated from a cDNA library constructed from germinatedMaackia amurensis seeds. From the nucleotide sequence, MAH was predicted to consist of 285 amino acid residues containing a signal peptide of 29 amino acids. The results also confirmed our previous findings from the amino acid sequence analysis, which indicated that two highly conserved amino acid residues in all other well-known leguminous lectins were replaced in MAH. These residues were lysine-105 and aspartic acid-135. The corresponding amino acid residues in other leguminous lectins were glycine and asparagine, respectively. These differences were due to the presence of nucleotides AAA and GAT in place of AAT/C and GGA/T.Abbreviations MAH Maackia amurensis haemagglutinin.     

Overexpression of genes of an extreme thermophile Thermus thermophilus, in Escherichia coli cells.

The 3-isopropylmalate dehydrogenase gene from an extreme thermophile, Thermus thermophilus, was not expressed in Escherichia coli unless a palindromic structure around the ribosome binding site was eliminated or a leader open reading frame was introduced into the upstream flanking region of the gene. This report suggests a way to increase the expression of this gene, with a high G+C content, in E. coli.     

Two kinds of thick filament in smooth muscle cells in the adductor of a clam, Chlamys nobilis

The ultrastructure of the opaque portion of the adductor muscle in the pecten Chlamys nobilis was investigated. The opaque portion was composed of smooth muscle cells that contained thin and thick filaments. The thick filaments were classified into two kinds, thinner and thicker, according to the statistical analysis of diameters. They were also classified as being shorter and longer, when isolated native filaments were examined. The thick filaments may consequently be classified into two kinds: thinner and shorter filaments, and thicker and longer ones. The thinner and shorter filaments were about 26.5 nm in diameter and 7.5 mum in length, and the thicker and longer ones were about 42.0 nm in diameter and 13.0 mum in length, respectively. A regular periodicity was apparent on the surface of the core after removal of myosin molecules from its surface. The periodicity seemed similar for the two kinds of thick filament.     

Nitric Oxide Reversibly Suppresses Xanthine Oxidase Activity

The effects of nitric oxide (NO) on xanthine oxidase (XOD) activity and the site(s) of the redox center(s) affected were investigated. XOD activity was determined by superoxide (O₂^-) generation and uric acid formation. NO reversibly and dose-dependently suppressed XOD activity in both determination methods. The suppression interval also disclosed a dose-dependent prolongation. The suppression occurred irrespective of the presence or absence of xanthine; indicating that the reaction product of NO and O₂^-, peroxynitrite, is not responsible for the suppression. Application of synthesized peroxynitrite did not affect XOD activity up to 2 μM. Methylene blue, which is an electron acceptor from Fe/S center, prevented the NO-induced inactivation. The results indicate that NO suppresses XOD activity through reversible alteration of the flavin prosthetic site.