Identification of a Gene Essential for Sheathed Structure Formation in Sphaerotilus natans, a Filamentous Sheathed Bacterium

Sphaerotilus natans, a filamentous bacterium that causes bulking in activated sludge processes, can assume two distinct morphologies, depending on the substrate concentration for growth; in substrate-rich media it grows as single rod-shaped cells, whereas in substrate-limited media it grows as filaments. To identify genes responsible for sheath formation, we carried out transposon Tn5 mutagenesis. Of the approximately 20,000 mutants obtained, 7 did not form sheathed structures. Sequencing of the Tn5-flanking regions showed that five of the seven Tn5 insertions converged at the same open reading frame, designated sthA. The deduced amino acids encoded by sthA were found to be homologous to glycosyltransferase, which is known to be involved in linking sugars to lipid carriers during bacterial exopolysaccharide biosynthesis. Disruption of the gene of the wild-type strain by inserting a kanamycin resistance gene cassette also resulted in sheathless growth under either type of nutrient condition. These findings indicate that sthA is a crucial component responsible for sheath formation.     

DOES ELASTIN CONTRIBUTE TO THE PERSISTENCE OF CORPORA ALBICANTIA IN THE OVARY OF THE COMMON DOLPHIN (DELPHINUS DELPHIS)

The corpora albicantia (CAs) from the ovaries of 39 common dolphins ( Delphinus delphis ) caught in driving fisheries were used, and histologically and immunohistochemically examined. The area of each corpus albicans (CA) and the proportion of that area occupied by elastin were measured using NIH-image software. In all CAs, elastoid material (EM) was apparent although EM area varied in each CA. CAs increased in number with dolphin age. Smaller CAs contained a higher proportion of EM. EM was completely digested by elastase, but not by collagenase. Furthermore, EM was immunostained with anti-α-elastin antibody. These results demonstrated that EM was elastin. The present study is the first to describe the presence of elastin in cetacean CAs. The higher proportion of elastin in small-sized CAs of common dolphins is suggested as the likely cause of the persistence of CA.     

A comprehensive analysis of allelic methylation status of CpG islands on human chromosome 11q: comparison with chromosome 21q.

It was generally believed that autosomal CpG islands (CGIs) escape methylation. However, our comprehensive analysis of allelic methylation status of 149 CGIs on human chromosome 21q revealed that a sizable fraction of them are methylated on both alleles even in normal blood cells. Here, we performed a similar analysis of 656 CGIs on chromosome 11q, which is gene-rich in contrast with 21q. The results indicate that 11q contains less methylated CGIs, especially those with tandem repeats and those in the coding or 3'-untranslated regions (UTRs), than 21q. Thus, methylation status of CGIs may substantially differ from one chromosome to another.     

Whole cell segmentation in solid tissue sections.

BACKGROUND: Understanding the cellular and molecular basis of tissue development and function requires analysis of individual cells while in their tissue context. METHODS: We developed software to find the optimum border around each cell (segmentation) from two-dimensional microscopic images of intact tissue. Samples were labeled with a fluorescent cell surface marker so that cell borders were brighter than elsewhere. The optimum border around each cell was defined as the border with an average intensity per unit length greater that any other possible border around that cell, and was calculated using the gray-weighted distance transform. Algorithm initiation requiring the user to mark two points per cell, one approximately in the center and the other on the border, ensured virtually 100% correct segmentation. Thereafter segmentation was automatic. RESULTS: The method was highly robust, because intermittent labeling of the cell borders, diffuse borders, and spurious signals away from the border do not significantly affect the optimum path. Computer-generated cells with increasing levels of added noise showed that the approach was accurate provided the cell could be detected visually. CONCLUSIONS: We have developed a highly robust algorithm for segmenting images of surface-labeled cells, enabling accurate and quantitative analysis of individual cells in tissue.     

Coexistence of two mitochondrial DNA haplotypes in Japanese populations of Hypera postica (Col., Curculionidae)

Abstract:  In Japan, the alfalfa weevil, Hypera postica , was first recorded in 1982 from Fukuoka and Okinawa Prefectures and has been spreading to many other prefectures. The weevil seriously infests the Chinese milk vetch, Astragalus sinicus , one of the most important honey resources for honeybees in Japan. Direct sequencing of partial mitochondrial DNA and PCR-RFLP data for alfalfa weevil individuals indicated the coexistence of two haplotypes at various localities in Japan. Molecular phylogenetic analysis for H. postica haplotypes and strains indicated that the two Japanese haplotypes had not derived from a single genetic origin. Based on the results, special comments are made on biological control measures using introduced parasitic waSPS.     

Interleukin-15 effectively potentiates the in vitro tumor-specific activity and proliferation of peripheral blood γδT cells isolated from glioblastoma patients

γδT cells play a regulatory role in both primary and metastatic tumor growth in humans. The mechanisms responsible for the activation and proliferation of circulating γδT cells should be fully understood prior to their adoptive transfer to cancer patients. We have examined in vitro functional effects of interleukin-15 (IL-15) on highly purified γδT cells isolated from glioblastoma patients. γδT cells constitutively express the heterotrimeric IL-2 receptor (IL-2R) αβγ, but the levels of IL-2Rβ or γ expression were not increased by incubation with saturating amounts of IL-15. IL-15 was shown to induce a maximal γδT cell proliferation, although at much higher concentrations (at least 2000 U/ml) than IL-2 (100 U/ml). Submaximal concentrations of IL-15 plus low concentrations of IL-2 produced an additive proliferative response. In contrast to the IL-2-induced response, this activity was completely or partially abrogated by anti-IL-2Rβ, or anti-IL-2Rγ antibodies, but not by anti-IL-2Rα antibodies. Incubation of γδT cells in the presence of IL-15 resulted not only in the appearance of NK and LAK activity, but also in specific autologous tumor cell killing activity, an additive effect being seen with IL-15 and IL-2. This IL-15-induced tumor-specific activity could be significantly blocked by anti-IL-2Rγ and anti-IL-2R-β mAb, but not by anti-IL-2Rα mAb. Thus, in contrast to IL-2, IL-15 activates tumor-specific γδT cells through the components of IL-2Rβ and IL-2Rγ, but not IL-2Rα. These enhanced in vitro tumor-specific and proliferative responses of γδT cells seen with IL-15 suggest a rational adjuvant imunotherapeutic use of γδT cells in cancer patients. Received: 23 January 1998 / Accepted: 20 May 1998     

In Vitro Susceptibility of Chlamydia pecorum to Macrolides,Tetracyclines, Quinolones and β-Lactam

The in vitro susceptibility of Chlamydia pecorum to two macrolides (clarithromycin and erythromycin), two tetracyclines (doxycycline and minocycline), two quinolones (ofloxacin and ciprofloxacin) and one β-lactam (ampicillin) was determined. The MICs were 0.004 to 0.008 μg/ml for clarithromycin, 0.008 to 0.031 μg/ml for doxycycline and minocycline, 0.063 to 0.125 μg/ml for erythromycin, 0.25 to 0.5 μg/ml for ofloxacin and 0.25 to 1.0 μg/ml for ciprofloxacin. The MIC for ampicillin was greater than 1,024 μg/ml. The results show clarithromycin and doxycycline are the two most effective drugs against C. pecorum.     

Analysis of Genetic Relationships and Antimicrobial Susceptibility of Verotoxin-Producing Escherichia coli Strains Isolated in Nagasaki Prefecture,Japan in 1996

A total of 19 Escherichia coli O157 isolates were obtained in Nagasaki Prefecture, in the southwestern part of Japan, between 1990 and 1996. Pulsed-field gel electrophoresis (PFGE) and computer-assisted analysis were applied to determine genetic relationships among these strains. Fragment patterns of the isolates in Nagasaki, as determined by PFGE, were compared with those of isolates in other areas where large outbreaks and sporadic cases of E. coli O157 infection occurred. Similarity values of all the strains isolated in Nagasaki Prefecture were over 0.65 except for E. coli O26. Some strains were identical to the strains isolated from the areas where large outbreaks occurred. All strains were susceptible to ampicillin, fosfomycin, minocycline, amikacin, ofloxacin and sulfamethoxazole-trimethoprim.     

Anti-Candida Activity of Cal protect in in Combination with Neutrophils or Lactoferrin

The effect of an anti-microbial protein, calprotectin, in combination with neutrophils on the growth of Candida albicans was investigated. The growth inhibition of C. albicans by murine neutrophils was augmented by the addition of a low concentration of calprotectin prepared from rat peritoneal exudate cells. The concentrations of calprotectin causing 50% inhibition of growth of C. albicans in the absence or presence of neutrophils at an effector-to-target (E/T) ratio of 30 and 60 were estimated to be 0.45, 0.34 and 0.28 U/ml, respectively. The anti-Candida activity of calprotectin was completely inhibited by 2 μM of zinc ion, while it only partially lowered the activity of the combination of calprotectin and neutrophils. Lactoferrin, which is an anti-microbial protein released from neutrophils, strongly inhibited the growth of C. albicans in combination with calprotectin. These results suggest that calprotectin and lactoferrin released from neutrophils may cooperate to inhibit the growth of C. albicans at a local lesion of the infection where there is an accumulation of neutrophils.