A monoclonal antibody to the carbohydrate chain on human hepatocellular carcinoma-associated antigen which suppressed tumor growth in nude mice

Summary There have been few reports stating that monoclonal antibody alone inhibits human solid tumor growth in vivo. The present study demonstrated that monoclonal antibody S1 (IgG2a), which recognized the antigenic determinant of the carbohydrate moiety, showed antibody-dependent cell (or macrophage)-mediated cytotoxicity (ADCC or ADMC) in conjunction with murine splenocytes of both BALB/c and athymic mice. In vivo experiments demonstrated that the antibody S1 clearly prolonged the survival of athymic mice which had been inoculated with a human liver carcinoma cell line. In addition, the antibody S1 significantly suppressed the human hepatoma line transplanted s.c. into nude mice. ¹²⁵I-Labeled monoclonal antibody S1 revealed that the antibody accumulated significantly in the tumor mass. Many mononuclear cells were observed surrounding tumor cells when the antibody was given. This model system might be useful for analyzing the ADCC (or ADMC) mechanism in vivo.     

Isolation of virus causing hemorrhagic fever with renal syndrome (HFRS) through a cell culture system

Twenty-three rat lung specimens collected in outbreaks of hemorrhagic fever with renal syndrome (HFRS) in three medical institutions were inoculated onto the VERO-E6 cell monolayers. After several blind passages, an agent growing serially in the cell cultures and reacting specifically with known HFRS-positive sera was isolated from two of these specimens. The two isolates were antigenically identical each other. The agent, named strain SR-11, was identified as the causative virus of HFRS by its antigenic identity with E6 cell-adapted HFRS virus, Hantaan 76-118 strain, and the specific reactions with sera from various HFRS cases.     

Identification of actin filaments in the rhabdomeral microvilli of Drosophila photoreceptors

The phototransductive microvilli of arthropod photoreceptors each contain an axial cytoskeleton. The present study shows that actin filaments are a component of this cytoskeleton in Drosophila. Firstly, actin was detected in the rhabdomeral microvilli and in the subrhabdomeral cytoplasm by immunogold labeling with antiactin. Secondly, the rhabdomeres were labeled with phalloidin, indicating the presence of filamentous actin. Finally, the actin filaments were decorated with myosin subfragment-1. The characteristic arrowhead complex formed by subfragment-1 decoration points towards the base of the microvilli, so that the fast growing end of each filament is at the distal end of the microvillus, where it is embedded in a detergent-resistant cap. Each microvillus contains more than one actin filament. Decorated filaments extend the entire length of each microvillus and project into the subrhabdomeral cytoplasm. This organization is comparable to that of the actin filaments in intestinal brush border microvilli. Similar observations were made with the photoreceptor microvilli of the crayfish, Procambarus. Our results provide an indication as to how any myosin that is associated with the rhabdomeres might function.     

Diversity of the Photoreceptors and Spectral Opponency in the Compound Eye of the Golden Birdwing,Troides aeacus formosanus

The compound eye of the Golden Birdwing, Troides aeacus formosanus (Papilionidae, Lepidoptera), is furnished with three types of ommatidia, which are clearly different in pigmentation around the rhabdom. Each ommatidium contains nine photoreceptors, whose spectral sensitivities were analyzed electrophysiologically. We identified nine spectral types of photoreceptor with sensitivities peaking at 360 nm (UV), 390 nm (V), 440 nm (B), 510 nm (BG), 540 nm (sG), 550 nm (dG), 580 nm (O), 610 nm (R), and 630 nm (dR) respectively. The spectral sensitivities of the V, O, R and dR receptors did not match the predicted spectra of any visual pigments, but with the filtering effects of the pigments around the rhabdom, they can be reasonably explained. In some of the receptors, negative-going responses were observed when they were stimulated at certain wavelengths, indicating antagonistic interactions between photoreceptors.     

Study of color variation in the solitary ascidian Halocynthia roretzi, collected in the Inland Sea of Japan

In the vicinity of Yashiro Island in the Inland Sea of Japan, the solitary ascidian (tunicate) Halocynthia roretzi with tunics of various colors were collected. Samples of these animals were sorted into three groups on the basis of visual observation of tunic color. The red group includes animals with dark-red, light-red, or orange tunics. The pink group includes animals with tunic colors ranging between red and white. The white group includes only animals with completely white tunics. Animals in the white group lacked color internally, with the exception of the hepatopancreas and the gonads in breeding season; the epidermis and gill basket were white. In contrast, animals of both the red group and the pink group were colored internally, with red-orange epidermis and yellow gill basket. Alloreactivity was tested by mixed-hemocyte incubation between different animals belonging to the same color group and between animals belonging to different color groups. Alloreactivity between animals of the white group was 56.3%, between animals of the pink group was 60.0%, and between animals of the red group was 69.3%. The relatively high frequency of compatible combinations among the white animals is discussed.     

Solid-Phase Artificial Chaperone-Assisted Refolding using Insoluble β-Cyclodextrin–Acrylamide Copolymer Beads

Solid-phase refolding methods are advantageous since they facilitate both separation of solid additives from the refolded protein and recycling of the additives. -Cyclodextrin–acrylamide copolymer hydrogel beads were used as a matrix for detergents in solid-phase artificial chaperone-assisted refolding and improved the yield of lysozyme (up to 65%) and carbonic anhydrase B (up to 80%), compared with conventional solid host matrices.Revisions received 29 September 2004     

Evaluation of surface hydrophobicity of immobilized protein with a surface plasmon resonance sensor

Immobilization is widely used to isolate agglutinative and associative proteins with large hydrophobic surfaces. Surface hydrophobicities of immobilized proteins were quantified by measuring the adsorption amounts of Triton X-100 as a hydrophobic probe with a biosensor that utilizes the phenomena of surface plasmon resonance (SPR). We measured SPR signal changes derived from adsorption of Triton X-100 to five kinds proteins and calculated the monolayer adsorption capacity using the Brunauer-Emmett-Teller equation, partly modified with a term for correcting an influence of the net charge of immobilized protein. SPR signal changes obtained by this method correlated with the values of surface hydrophobicities obtained by conventional assay using a hydrophobic probe. Thus this measuring method using an SPR sensor and Triton X-100 is expected to be a tool for quantifying surface hydrophobicities of immobilized proteins.     

Hantavirus-specific CD8(+)-T-cell responses in newborn mice persistently infected with Hantaan virus

The relationship between virus-specific CD8(+)-T-cell responses and viral persistence was studied in mice by using Hantaan virus (HTNV). We first established a simple method for measuring levels of virus-specific CD8(+) T cells by flow cytometry. Next, to produce a mouse model of persistent HTNV infection, newborn mice were inoculated subcutaneously within 24 h of birth with 1 or 0.1 50% newborn mouse lethal dose of HTNV. All mice that escaped lethal infection were persistently infected with HTNV until at least 30 days after virus inoculation and had no virus-specific CD8(+) T cells producing gamma interferon (IFN-gamma). Subsequently, the virus was eliminated from some of the mice, depending on the appearance of functional virus-specific CD8(+) T cells, which have the ability to produce IFN-gamma and tumor necrosis factor alpha (TNF-alpha) and have cytotoxic activity. Neutralizing antibodies were detected in all mice, regardless of the presence or absence of virus. In the acute phase, which occurs within 30 days of infection, IFN-gamma-producing HTNV-specific CD8(+) T cells were detected on day 15 after virus inoculation. However, TNF-alpha production and the cytotoxic activity of these specific CD8(+) T cells were impaired and HTNV was not removed. Almost all of these specific CD8(+) T cells disappeared by day 18. These results suggest that functional HTNV-specific CD8(+) T cells are important for clearance of HTNV.     

Levels and behavior of 2-phenylbenzotoriazole-type mutagens in the effluent of a sewage treatment plant

We previously reported on the isolation and structural determination of five 2-phenylbenzotriazole (PBTA)-type mutagens (PBTA-1, PBTA-2, PBTA-3, PBTA-4 and PBTA-6) in blue rayon/cotton adsorbed substances collected from surface waters at sites located downstream of sewage treatment plants. We also noted that PBTA-1 and PBTA-2 were discharged from sewage treatment plants and subsequently diluted or decomposed while moving down the Yodo River system. However, it has not been investigated whether they are commonly discharged from sewage treatment plants into rivers. The main purpose of this study was to make a comprehensive survey of levels and behavior of PBTA-type mutagens in effluents discharged from the sewage treatment plant located along the bank of the Uji River, one tributary of the Yodo River system. Water samples were collected at the outlet of the sewage treatment plant for 16 consecutive days in May 1999 and 11 consecutive days in December 1999. Organic constituents were obtained via sorption to blue rayon and subsequent methanol elution. Extract mutagenic activity was measured using Salmonella typhimurium YG1024 with metabolic activation. PBTA-type mutagens (PBTA-1, PBTA-2, PBTA-3, PBTA-4, PBTA-5 and PBTA-6) were quantified by HPLC with electrochemical detection, followed by HPLC purification on reverse-phase columns. The study showed that PBTA-2, PBTA-3, PBTA-4 and PBTA-6 were detected in most samples. The total contribution of these four PBTA-type mutagens to overall extract mutagenicity is on average 33% for the May 1999 sample and 58% for the December 1999 sample. The individual PBTA compounds that had the largest contribution to the overall mutagenicity were PBTA-3 and PBTA-4, accounting for 11 and 16% in May 1999, and 25 and 26% in December 1999. A further comparative study was done in December 1999 using the blue rayon hanging method and the results were similar to those obtained using the blue rayon column method. In conclusion, the present study showed that PBTA-2, PBTA-3, PBTA-4 and PBTA-6 were commonly discharged from a sewage treatment plant into the Uji River, and they accounted for a substantial portion of the effluent mutagenicity.     

Physiological role of soluble fumarate reductase in redox balancing during anaerobiosis in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, there are two isoenzymes of fumarate reductase (FRDS1 and FRDS2), encoded by the FRDS and OSM1 genes, respectively. Simultaneous disruption of these two genes results in a growth defect of the yeast under anaerobic conditions, while disruption of the OSM1 gene causes slow growth. However, the metabolic role of these isoenzymes has been unclear until now. In the present study, we found that the anaerobic growth of the strain disrupted for both the FRDS and OSM1 genes was fully restored by adding the oxidized form of methylene blue or phenazine methosulfate, which non-enzymatically oxidize cellular NADH to NAD(+). When methylene blue was added at growth-limiting concentrations, growth was completely arrested after exhaustion of oxidized methylene blue. In the double-disrupted strain, the accumulation of succinate in the supernatant was markedly decreased during anaerobic growth in the presence of methylene blue. These results suggest that fumarate reductase isoenzymes are required for the reoxidation of intracellular NADH under anaerobic conditions, but not aerobic conditions.