Measurement of sulfidopeptide leukotrienes and their metabolism in human synovial fluid of patients with rheumatoid arthritis

Leukotriene (LT)C4 in the synovial fluid of patients with osteoarthritis deformans (OA) and rheumatoid arthritis (RA) was measured by radioimmunoassay (RIA) after extraction with Sep-Pak C18 cartridge. The amounts of immunoreactive LTC4 (i-LTC4) in samples from patients with OA and RA were not significantly different, being 0.198 +/- 0.018 pmol/ml (n = 11) and 0.179 +/- 0.016 pmol/ml (n = 12), respectively. After separation by high performance liquid chromatography (HPLC) and measurement by RIA, the levels of other sulfidopeptide LTs, such as LTD4 and LTE4, in synovial fluid from patients with RA were found to be significantly higher than those in fluid from patients with OA. The leukocyte number in synovial fluids did not correlate with the i-LTC4 level. The metabolic activities of these synovial fluids were determined by incubating them with 3H-LTC4 and then separating sulfidopeptide LTs by HPLC. The conversion of LTC4 to LTD4 in synovial fluids of patients with OA and RA were similar, but the dipeptidase activity converting LTD4 to LTE4 was higher in fluid from patients with RA. It is suggested that a high level of LTE4 may contribute to exudation of synovial fluid, since LTE4 increases vascular permeability.     

Nicotine induces dendritic spine remodeling in cultured hippocampal neurons

Cholinergic neurons in the CNS are involved in synaptic plasticity and cognition. Both muscarinic and nicotinic acetylcholine receptors (nAChRs) influence plasticity and cognitive function. The mechanism underlying nAChR‐induced plasticity, however, has remained elusive. Here, we demonstrate morphological changes in dendritic spines following activation of α4β2* nAChRs, which are expressed on glutamatergic pre‐synaptic termini of cultured hippocampal neurons. Exposure of the neurons to nicotine resulted in a lateral enlargement of spine heads. This was abolished by dihydro‐β‐erythroidine, an antagonist of α4β2* nAChRs, but not by α‐bungarotoxin, an antagonist of α7 nAChRs. Tetanus toxin or a mixture of 2‐amino‐5‐phosphonovaleric acid and 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione, antagonists of NMDA‐ and AMPA‐type glutamate receptors, blocked the nicotine‐induced spine remodeling. In addition, nicotine exerted full spine‐enlarging response in the post‐synaptic neuron whose β2 nAChR expression was knocked down. Finally, pre‐treatment with nicotine enhanced the Ca²⁺‐response of the neurons to glutamate. These data suggest that nicotine influences the activity of glutamatergic neurotransmission through the activation of pre‐synaptic α4β2 nAChRs, resulting in the modulation of spinal architecture and responsiveness. The present findings may represent one of the cellular mechanisms underlying cholinergic tuning of brain function.

     

Disparate effects of monensin and colchicine on intracellular processing of secretory proteins in cultured rat hepatocytes

We have studied the biosynthesis and intracellular processing of three major secretory proteins, albumin, alpha 1-protease inhibitor and alpha 2u-globulin, in cultured rat hepatocytes. The effect of secretion-blocking agents, monensin, a monovalent ionophore, and the microtubule-affecting agents colchicine and taxol was determined. In the control cells, alpha 1-protease inhibitor, a glycoprotein, was first synthesized as an endoglycosidase-H-sensitive form with Mr 51 000, and then processed to two endoglycosidase-H-resistant forms having Mr 51 000 and 56 000, the latter of which was secreted into the medium. Initially synthesized proalbumin was converted with chase to serum-type albumin, while no pro-type precursor was identified for alpha 2u-globulin. In the cells treated with colchicine or taxol, in which secretion was greatly inhibited, the fully processed alpha 1-protease inhibitor and albumin accumulated and were finally secreted into the medium. In the monensin-treated cells, however, most of the newly synthesized alpha 1-protease inhibitor and albumin were not processed to the final mature forms, resulting in accumulation of two 51 000-Mr forms and proalbumin, respectively. Moreover in treated cells, proalbumin and the endoglycosidase-H-resistant alpha 1-protease inhibitor were finally secreted into the medium. Such an effect was not caused by NH4Cl which also inhibited the secretion and is known to exert the similar effect as monensin on the receptor-mediated endocytosis pathway. Based on these results, the use of monensin may prove valuable for more detailed analysis of intracellular processing of various proteins.     

Crystal structure of tobacco PR-5d protein at 1.8 A resolution reveals a conserved acidic cleft structure in antifungal thaumatin-like proteins

The crystal structure of tobacco PR-5d, an antifungal thaumatin-like protein isolated from cultured tobacco cells, was determined at the resolution of 1.8 A. The structure consists of 208 amino acid residues and 89 water molecules with a crystallographic R-factor of 0.169. The model has good stereochemistry, with respective root-mean-square deviations from the ideal values for bond and angle distances of 0.007 A and 1.542 degrees. Of the homologous PR-5 proteins, only those with antifungal activity had a common motif, a negatively charged surface cleft. This cleft is at the boundary between domains I and II, with a bottom part consisting of a three-stranded antiparallel beta-sheet in domain I. The acidic residues located in the hollow of the cleft form the beta-sheet region. Sequence and secondary structure analyses showed that the amino acid residues comprising the acidic cleft of PR-5d are conserved among other antifungal PR-5 proteins. This is the first report on the high-resolution crystal structure of an antifungal PR-5 protein. This structure provides insight into the function of pathogenesis-related proteins.     

Catalytic residues and substrate specificity of scytalidoglutamic peptidase, the first member of the eqolisin in family (G1) of peptidases

Scytalidoglutamic peptidase (SGP) is the first-discovered member of the eqolisin family of peptidases with a unique structure and a presumed novel catalytic dyad (E136 and Q53) [Fujinaga et al., PNAS 101 (2004) 3364-3369]. Mutants of SGP, E136A, Q53A, and Q53E lost both the autoprocessing and enzymatic activities of the wild-type enzyme. Coupled with the results from the structural analysis of SGP, Glu136 and Gln53 were identified as the catalytic residues. The substrate specificity of SGP is unique, particularly, in the preference at the P3 (basic amino acid), P1' (small a.a.), and P3' (basic a.a.) positions. Superior substrates and inhibitors have been synthesized for kinetic studies based on the results reported here. kcat, Km, and kcat/Km of SGP for D-Dap(MeNHBz)-GFKFF*ALRK(Dnp)-D-R-D-R were 34.8 s-1, 0.065 microM, and 535 microM-1 s-1, respectively. Ki of Ac-FKF-(3S,4S)-phenylstatinyl-LR-NH2 for SGP was 1.2x10(-10) M. Taken together, we can conclude that SGP has not only structural and catalytic novelties but also a unique subsite structure.     

Spatiotemporal analysis of Ca(2+) waves in relation to the sperm entry site and animal-vegetal axis during Ca(2+) oscillations in fertilized mouse eggs

Fertilized mouse eggs exhibit repetitive rises in intracellular Ca(2+) concentration ([Ca(2+)](i)) necessary for egg activation. Precise spatiotemporal dynamics of each [Ca(2+)](i) rise were investigated by high-speed Ca(2+) imaging during early development of monospermic eggs. Every [Ca(2+)](i) rise involved a Ca(2+) wave. In the first Ca(2+) transient, [Ca(2+)](i) increased in two steps separated by a "shoulder" point, suggesting two distinct Ca(2+) release mechanisms. The first step was a Ca(2+) wave that propagated from the sperm-fusion site to its antipode in 4-5 s (velocity, approximately 20 microm/s in most eggs). The second step from the shoulder to the peak was a nearly uniform [Ca(2+)](i) rise of 12-15 s. A slight cytoplasmic movement followed the Ca(2+) wave in the same direction and recovered in 25-35 s. These characteristics changed as follows, as Ca(2+) oscillations progressed during the second meiosis up to their cessation at the stage of pronuclei formation ( approximately 3 h after fertilization). (1) The duration of Ca(2+) transients became shorter. (2) The shoulder point shifted to higher levels and the first step occupied most of the rising phase. (3) The rate of [Ca(2+)](i) rise became greater and wave speeds increased up to 80-100 microm/s or more. (4) The transient cytoplasmic movement always resulted from the Ca(2+) wave, although its displacement became smaller. (5) The Ca(2+) wave initiation site was freed from the sperm-fusion or -entry site and eventually localized in the cortex of the vegetal hemisphere. Since the shift of the wave initiation site to the vegetal cortex is observed in fertilized eggs of nemertean worms and ascidians, this might be an evolutionarily conserved feature.     

Intraspecific divergence of Saccharomyces kluyveri as revealed by the nucleotide sequences of 18S-28S rRNA spacer regions and alpha-galactosidase MEL genes

In the five strains classified as the yeast Saccharomyces kluyveri, several substitutions were observed in the two internal transcribed spacer regions between 18S and 28S rRNA. A PCR reaction with primers targeted to the MEL1 gene of Saccharomyces cerevisiae amplified fragments of the expected size, and those sequences showed significant divergence in the strains of S. kluyveri.