Induction of apoptosis by carboplatin and hyperthermia alone or combined in WERI human retinoblastoma cells.

This paper investigated the induction of apoptosis and perturbation of cell cycle progression caused by carboplatin (CPt) and hyperthermia alone or combined in WERI human retinoblastoma cells in vitro. An incubation of the cells with 25 or 50 microm of CPt at 37 degrees C caused apoptosis, which progressively increased during the 24-72 h treatment. Hyperthermia at 42.5 degrees C for 1 h induced apoptosis, which became significant from 24 h after the heating. Heating the cells in the presence of CPt and subsequent incubation with CPt was far more effective than treating the cells with hyperthermia or CPt treatment alone in inducing apoptosis in the WERI cells, indicating that the combination of these two modalities is potentially useful for the treatment of retinoblastoma. It appeared that the apoptosis in WERI cells caused by hyperthermia and CPt occurs during G1 phase. An interesting observation was that caspase 9 activation preceded the release of cytochrome C from mitochondria during apoptosis in WERI cells, contrary to the general notion that caspase 9 is activated by cytochrome C.

Neutralization epitopes on the antigenic domain II of the Orientia tsutsugamushi 56-kDa protein revealed by monoclonal antibodies.

Monoclonal antibodies (MoAbs) reactive with the authentic Orientia tsutsugamushi 56-kDa protein were generated. MoAb FS10 and FS15 showed in vitro, as well as, in vivo neutralizing activity upon O. tsutsugamushi infection. Deletion mutants of the gene for 56-kDa protein of O. tsutsugamushi Boryong were expressed to map the binding region. FS10 and FS15 are bound to amino acids (aa) located in an antigenic domain II, at residues 140-160 and 187-214, respectively. Computer modeling indicated that aa 146-153 were important for antigenicity against FS10. A sequence for aa 142-150 was highly homologous between oriential strains. These results suggest that the antigenic determinant for neutralizing MoAbs is an epitope within aa 140-160. Furthermore, this region may be important for the adhesion/invasion or intracellular survival of O. tsutsugamushi within host cells.

Apoptosis and cell cycle progression in an acidic environment after irradiation.

Apoptosis and cell cycle progression in HL60 cells irradiated in an acidic environment were investigated. Apoptosis was determined by TUNEL staining, PARP cleavage, DNA fragmentation, and flow cytometry. The majority of the apoptosis that occurred in HL60 cells after 4 Gy irradiation took place after G(2)/M-phase arrest. When irradiated with 12 Gy, a fraction of the cells underwent apoptosis in G(1) and S phases while the rest of the cells underwent apoptosis in G(2)/M phase. The apoptosis caused by 4 and 12 Gy irradiation was transiently suppressed in medium at pH 7.1 or lower. An acidic environment was found to perturb progression of irradiated cells through the cell cycle, including progression through G(2)/ M phase. Thus it was concluded that the suppression of apoptosis in the cells after 4-12 Gy irradiation in acidic medium was due at least in part to a delay in cell cycle progression, particularly the prolongation of G(2)/M-phase arrest. Irradiation with 20 Gy indiscriminately caused apoptosis in all cell cycle phases, i.e. G(1), S and G(2)/M phases, rapidly in neutral pH medium and relatively slowly in acidic pH medium. The delay in apoptosis in acidic medium after 20 Gy irradiation appeared to result from mechanisms other than prolonged G(2)/ M-phase arrest.

Disassembly of focal adhesions during apoptosis of endothelial cell line ECV304 infected with Orientia tsutsugamushi.

Many bacterial pathogens induce apoptosis in their host cells. We observed the cellular effect of ECV304 cells infected with Orientia tsutsugamushi. The infected cells became rounded and floated in culture supernatant. These floating cells as well as adherent cells exhibited typical features of apoptosis, such as DNA fragmentation and TUNEL staining. As many cells detached from growth substrate, we examined the focal adhesion using the immunofluorescence assay method and observed decreased focal adhesions in heavily infected cells. As endothelial cells could undergo apoptosis by the loss of focal adhesions, this change of focal adhesions may account for the Orientia-induced apoptosis.

Apoptosis of endothelial cell line ECV304 persistently infected with Orientia tsutsugamushi.

Endothelial cells are major targets of Orientia tsutsugamushi. To examine the consequences of the infection of endothelial cells with O. tsutsugamushi, we used human endothelial cell line ECV304. Persistent infection was established and infected cultures could be maintained for over seven months without the addition of normal cells. The heavily infected cells became round and floated in the culture medium, harboring large numbers of organisms inside them. Some of the infected ECV304 cells showed features of apoptotic cells, as determined by the terminal deoxytransferase-mediated dUTP nick end-labeling reaction and DNA fragmentation. We also found that O. tsutsugamushi increased transcription of the mRNAs of proinflammatory cytokines such as IL-6 and IL-8. These results show the first evidence of in vitro-persistent infection by O. tsutsugamushi, which may be related to in vivo persistence reported previously.

Pathologic study of mice infected with Rickettsia tsutsugamushi R19 strain.

Scrub typhus, an acute febrile infectious disease caused by R. tsutsugamushi, has been reported from various parts of the far east and pacific rim of Asia including Korea. It is well known that all human pathogenic rickettsia share an affinity to endothelial cells of the small blood vessels and evoke vascular inflammation variably associated with a rash, microthrombi, and hemorrhage. We infected the ICR mice by inoculating sublethal doses of R. tsutsugamushi R19 strain intraperitoneally and observed the pathologic changes by time sequence. The histopathologic features of experimentally induced scrub typhus in the mice were generally nonspecific interstitial inflammations characterized by interstitial pneumonitis, periportal inflammation, multifocal hepatic necrosis, interstitial nephritis, sinusoidal engorgement, and lymphohistiocytic cell infiltration in lymph nodes and spleen. Contrary to the general features of other rickettsial diseases, the pathologic process of scrub typhus experimentally induced by R. tsutsugamushi R19 strain mainly involved the interstitial connective tissue but not the blood vessels.

Distribution of HLA class I alleles and haplotypes in Korean.

The antigen (phenotype), gene (allele) and haplotype frequencies of HLA class I were analysed in 4,622 Koreans. With allele frequencies of over 0.05, the most frequent HLA-A,-B and -C antigens were A2, A24, A33, A11, A26, A31; B62, B51, B44, B54, B61, B35, B58, B60; Cw3, Cw1, Cw4, Cw7. Of these A2, A24, Cw1 and Cw3 were present in very high frequencies, respectively (0.3211, 0.2200, 0.2204, and 0.3737). The most common haplotypes with frequencies larger than 0.02 were A2-Blank, A33-B44, A33-B58, A11-B62, A24-B51, A24-B54, A2-B27, B54-Cw1, B58-Cw3, B51-Blank, B61-Cw3, B62-Cw4, B35-Cw3, B44-Blank, B60-Cw3, B27-Cw1, A2-Cw3, A2-Cw1, A24-Cw1, A33-Cw3, A26-Cw3, and A11-Cw4. A significant negative linkage disequilibrium was found for the haplotypes of A2-B7, A2-B44, A2-B58, A24-B13, A24-B27, A33-B54 and A33-B62, of which frequencies were larger than 0.003. The B-C and A-C haplotypes which showed the significant negative linkage disequilibrium were B44-Cw1, B51-Cw1, B44-Cw3,B62-Blank, A2-Cw4, A2-Blank, A11-Cw3, A11-Blank and A33-Cw1 and had frequencies higher than 0.01. The findings presented here could be used per se to estimate the populational relationships or as the control data for HLA-disease investigation. Furthermore they could provide the scope for the definition of new antigens.

Nucleotide sequence and coding capacity of the large (L) genomic RNA segment of Seoul 80-39 virus, a member of the hantavirus genus.

The nucleotide sequence of the large (L) genomic RNA segment of Seoul 80-39 virus was determined from overlapping cDNA clones. The virion L RNA segment is 6530 nucleotides long. The 3' and 5' terminal sequences are inversely complementary for 15 bases. The viral complementary-sense RNA contains a single open reading frame from an AUG codon at nucleotide position 37-39 to a UAA stop codon at nucleotide position 6490-6492. This ORF could encode a polypeptide of 2151 amino acids (246,662 kDa) which likely corresponds to the L protein detected in purified viral particles (Elliott et al., 1984) and is assumed to be an RNA-dependent RNA polymerase molecule (Schmaljohn and Dalrymple, 1983). Comparison of the L protein of the Seoul 80-39 virus with the polymerase proteins encoded by other negative-stranded RNA viruses revealed 44% similarity only with the part of the Bunyamwera virus L protein (Elliott, 1989) and a very weak homology with the PB1 protein of influenza virus.

Molecular characterization of the M genomic segment of the Seoul 80-39 virus; nucleotide and amino acid sequence comparisons with other hantaviruses reveal the evolutionary pathway.

The genomic M segment of Seoul 80-39 virus was characterized by cloning and nucleotide sequence analysis. The virion M RNA segment is 3651 nucleotides long with the 3' and 5' terminal sequences inversely complementary for 20 bases. A single open reading frame was detected in the viral complementary-sense RNA which can encode a polypeptide of 1133 amino acids. The Seoul 80-39 virus M segment was compared with the M segments of related viruses, Hantaan 76-118, Hallnas B1 and Sapporo Rat (SR-11) virus. Our results demonstrate a significant similarity between M RNA segments of the Seoul 80-39, Hantaan 76-118, Hallnas B1 and SR-11 viruses. The degree of conservation of both nucleic acid and protein sequences between these viruses reveals a close evolutionary relationship. Furthermore, it is evident that the serotypic profile of hantaviruses is determined by the rodent host species from which the virus was isolated and not by the geographical area.