Genetic typing of the Senescence-Accelerated Mouse (SAM) strains with microsatellite markers

The Senescence-Accelerated Mouse (SAM) strains constitute a murine model of accelerated senescence originating from the ancestral AKR/J strains and consist of nine senescence-prone (SAMP) strains and four senescence-resistant (SAMR) strains. The chromosomes (Chrs) of the SAM strains were typed with 581 microsatellite markers amplified by PCR, and the fundamental genetic information of the SAM strains was obtained. One-third of the examined markers displayed polymorphism among the strains, and only two alleles were detected in almost all loci among the SAM and AKR/J strains. However, in 12 loci (5.6% of total 215 polymorphic markers), the third allele was detected among the SAM strains. The genetic typing and developmental history suggested that the SAM strains were related inbred strains developed by the accidental crossing between the AKR/J strain and other unknown strain(s). Comparison of the distribution of the loci in the SAMP and the SAMR series revealed notable differences in the four regions on Chrs 4, 14, 16, and 17. This indicated that some of these chromosomal sites might contain the genes responsible for accelerated senescence in the SAMP series. Received: 17 July 1998 / Accepted: 17 November 1998     

Thiazolidinedione inhibits production of RANTES in a cytokine-treated human lung epithelial cell line.

The chemokine RANTES is a potent chemoattractant for eosinophils. RANTES is produced by lung epithelial cells during eosinophil-rich inflammatory diseases such as asthma. In this study, we examined the effects of thiazolidinediones (TZD) on RANTES expression in a human lung epithelial cell line, A549. In A549 cells, interleukin-1beta and tumor necrosis factor-alpha induced endogenous RANTES protein secretion, mRNA expression, and promoter activity. The TZD inhibited these effects. Our data indicate that the suppression of the expression of RANTES can be accomplished by TZD treatment, raising the possibility that TZD might be of therapeutic value in diseases such as asthma.     

Thiazolidinedione inhibits the production of monocyte chemoattractant protein-1 in cytokine-treated human vascular endothelial cells.

The chemokine monocyte chemoattractant protein-1 is a potent chemoattractant for monocytes. Monocyte chemoattractant protein-1 is produced by vascular endothelial cells during inflammatory diseases such as atherosclerosis. In this study, we examined the effects of a thiazolidinedione on monocyte chemoattractant protein-1 expression in human vascular endothelial cells. In human vascular endothelial cells, interleukin-1beta and tumor necrosis factor-alpha induced endogenous monocyte chemoattractant protein-1 protein secretion, mRNA expression and promoter activity. The thiazolidinedione inhibited these effects. In summary, our results indicated that the suppression of the expression of monocyte chemoattractant protein-1 can be accomplished by thiazolidinedione treatment, raising the possibility that thiazolidinedione may be of therapeutic value in the treatment of diseases such as atherosclerosis.     

Determination of Abundance and Biovolume of Bacteria in Sediments by Dual Staining with 4′,6-Diamidino-2-Phenylindole and Acridine Orange: Relationship to Dispersion Treatment and Sediment Characteristics

We measured the abundance and biovolume of bacteria in intertidal sediments from Tokyo Bay, Japan, by using a dual-staining technique (4′,6-diamidino-2-phenylindole and acridine orange) and several dispersion techniques (ultrasonic cleaner, ultrasonic sonicator, and tissue homogenizer). Dual staining reduced serious background fluorescence, particularly when used for silt-, clay-, and detritus-rich sediments, and allowed us to distinguish bacteria from other objects during both counting and sizing. Within the studied samples, the number of bacterial cells ranged from 0.20 × 10⁹ to 3.54 × 10⁹ g of wet sediment⁻¹. With the cleaner and sonicator treatments, the bacterial numbers for all of the sites initially increased with dispersion time and then became constant. For the homogenizer treatments, the highest bacterial numbers were observed with the shortest (0.5- to 2-min) treatments, and the counts then declined steeply as the homogenization time increased, indicating that cell destruction occurred. The cleaner treatment had the possibility of insufficient dispersion of bacteria for fine-grain sediments. Within the studied samples, the bacterial biovolume ranged from 0.07 to 0.22 μm³. With the cleaner and sonicator treatments, the biovolume peaked during the shorter dispersion time. This pattern was caused not by cell destruction but by the incremental portion of dispersed small cells. We concluded that with the cleaner and sonicator treatments, the longer dispersion time reflected the real size spectrum and was preferable for accurate estimation of mean bacterial biovolumes.     

Characterization of Serine Endopeptidases in Cotyledons of Germinated Vigna mungo Seeds

Vigna mungo seeds. SEP activity was separated into two isoforms by CM-cellulose column chromatography. These forms, termed SEP-1 and SEP-II, showed endopeptidase activities even at acidic pH, suggesting that SEPs are unique serine endopeptidases, since most serine proteases are optimum at neutral pH. Received 14 December 1998/ Accepted in revised form 22 February 1999     

Phosphorylation and/or Presence of Serine 37 in the Movement Protein of Tomato Mosaic Tobamovirus Is Essential for Intracellular Localization and Stability In Vivo

The P30 movement protein (MP) of tomato mosaic tobamovirus (ToMV) is synthesized in the early stages of infection and is phosphorylated in vivo. Here, we determined that serine 37 and serine 238 in the ToMV MP are sites of phosphorylation. MP mutants in which serine was replaced by alanine at positions 37 and 238 (LQ37A238A) or at position 37 only (LQ37A) were not phosphorylated, and mutant viruses did not infect tobacco or tomato plants. By contrast, mutation of serine 238 to alanine did not affect the infectivity of the virus (LQ238A). To investigate the subcellular localization of mutant MPs, we constructed viruses that expressed each mutant MP fused with the green fluorescent protein (GFP) of Aequorea victoria. Wild-type and mutant LQ238A MP fusion proteins showed distinct temporally regulated patterns of MP-GFP localization in protoplasts and formation of fluorescent ring-shaped infection sites on Nicotiana benthamiana. However mutant virus LQ37A MP-GFP did not show a distinct pattern of localization or formation of fluorescent rings. Pulse-chase experiments revealed that MP produced by mutant virus LQ37A was less stable than wild-type and LQ238A MPs. MP which contained threonine at position 37 was phosphorylated, but the stability of the MP in vivo was very low. These studies suggest that the presence of serine at position 37 or phosphorylation of serine 37 is essential for intracellular localization and stability of the MP, which is necessary for the protein to function.     

Antitubulin effects of aminobenzothiophene-substituted triethylated chromones

In the course of our continuing studies on the 2-(benzo[b]thiophene-3′-yl)-6,8,8-triethyldesmosdumotin B (TEDB-TB) series, we designed and synthesized nine amino-TEDB-TB derivatives to improve pharmaceutical properties, identify structure activity relationships, and discover novel antitubulin agents. Among all newly synthesized amino-TEDB-TBs, the 5′- and 6′-amino derivatives, 6 and 7, exhibited significant antiproliferative activity against five human tumor cell lines, including an MDR subline overexpressing P-gp. The IC₅₀ values of 0.50–1.01 µM were 3–6 times better than those of previously reported hydroxy-TEDB-TBs. Compounds 6 and 7 inhibited tubulin polymerization, induced both depolymerization of interphase microtubules and multiple spindle formations, and caused cell arrest at prometaphase. Among all compounds, compound 7 scored best pharmaceutically with LogP 2.11 and biologically with greater antiproliferative activity and induction of cell cycle arrest at prometaphase.     

Design,semisynthesis and potent cytotoxic activity of novel 10-fluorocamptothecin derivatives

Fluorination is a well-known strategy for improving the bioavailability of bioactive molecules in the lead optimization phase of drug discovery projects. In an attempt to improve the antitumor activity of camptothecins (CPTs), novel 10-fluoro-CPT derivatives were designed, synthesized and evaluated for cytotoxicity against five human cancer cell lines (A-549, MDA-MB-231, KB, KB-VIN and MCF-7). All of the derivatives showed more potent in vitro cytotoxic activity than the clinical CPT-derived drug irinotecan against the tumor cell lines tested, and most of them showed comparable or superior potency to topotecan. Remarkably, compounds 16b (IC₅₀, 67.0 nM) and 19b (IC₅₀, 99.2 nM) displayed the highest cytotoxicity against the multidrug-resistant (MDR) KB-VIN cell line and merit further development as preclinical drug candidates for treating cancer, including MDR phenotype. Our study suggested that incorporation of a fluorine atom into position 10 of CPT is an effective method for discovering new potent CPT derivatives.     

Obligate gut symbiotic association in the sloe bug <Emphasis Type="Italic">Dolycoris baccarum</Emphasis> (Hemiptera: Pentatomidae)

A number of phytophagous stinkbugs are associated with specific bacterial symbionts in their alimentary tracts. The sloe bug Dolycoris baccarum (Linnaeus), a notorious pest of diverse crops, possesses a number of sac-like tissues, called crypts, in a posterior section of the midgut, wherein a specific bacterial symbiont colonizes. Here we characterized the symbiotic bacterium of D. baccarum by histological analysis, molecular phylogeny, and diagnostic PCR with a specific primer set. The cloning and sequencing analyses of bacterial 16S rRNA genes and fluorescent in situ hybridization demonstrated that the sloe bug is associated with a single species of Gammaproteobacteria in the midgut crypts. Molecular phylogenetic analysis strongly suggested that the symbiont should be placed in the genus Pantoea of the Enterobacteriaceae. Diagnostic PCR and egg surface sterilization with formalin indicated the stinkbug vertically transmits the Pantoea symbiont via egg-smearing. The sterilization-produced aposymbiotic nymphs showed high mortality and no insects reached adulthood. In addition, the Pantoea symbiont was uncultivable outside the insect host, indicating an obligate and intimate host-symbiont association.