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We generated random Tn5 mutations in Synechocystis sp. PCC 6803 in search for genes involved in the signal transduction cascade for the cyanobacterial gliding motility. One of the non-gliding Tn5 mutants, S1-105, had an insertional inactivation in the slr1044 gene encoding a putative methyl-accepting chemotaxis protein. Interposon mutation on the slr1044 (named ctr1) in the bacterium also eliminated gliding motility. In the interposon mutant, the expression of pilA1 was 5-fold decreased compared with that of wild-type and thick pili, that are believed to be the motor for gliding, could not be observed by an electron microscope. Therefore, we suggest that the Ctr1 protein functions as a transducer that regulates the expression of pilA1, and thus is required for the biogenesis of thick pili.

This study was performed to investigate the effect of Puerariae Flos (PF) and Puerariae Radix (PR) water extracts on the activities and mRNA expression of three hepatic antioxidant enzymes in ethanol-treated rats. Male Sprague-Dawley rats were divided into four groups, a control, ethanol-treated, ethanol plus PF-treated, and ethanol plus PR-treated group with seven rats per group. Ethanol (25 % v/v, 5 g/kg body weight) was orally administered once a day for 5 weeks. The PF and PR water extracts were supplemented in a diet based on 1.2 g of raw PF or PR/kg body weight/day. Ethanol administration without the PF or PR supplement significantly lowered the activities of hepatic Cu/Zn SOD and catalase (CAT), whereas it increased the hepatic GSH-Px activity. However, the PF and PR supplementation resulted in a significant increase in the Cu/Zn SOD and/or CAT activities and a significant decrease in the GSH-Px activity in the ethanol-treated rats. The mRNA levels of these antioxidant enzymes in the ethanol-treated rats were normalized to the control level by the PF or PR supplement. The hepatic glutathione content, which was significantly lower in the ethanol-treated group than in the control group, was also normalized to the control level by supplementing with either PF or PR. The PF or PR supplement resulted in lowering the hepatic malondialdehyde to the control level in the ethanol-treated rats.

Background: Dieulafoy's lesion has unique endoscopic and histopathologic characteristics. This is a clinical trial of endoscopic therapy in 24 patients with Dieulafoy's lesions., Methods: Patients were divided into 2 groups according to initial endoscopic treatment method. Data were analyzed with respect to clinical and endoscopic characteristics as well as outcomes. The 24 patients were evenly divided into mechanical (9 hemoclipping, 3 band ligation) and injection groups (12)., Results: The average number of therapeutic endoscopic sessions needed to achieve permanent hemostasis for the mechanical and injection groups were 1.17 and 1.67, respectively. Initial hemostasis was achieved in 91.7% of patients undergoing mechanical therapy and 75% of those undergoing injection therapy, with none in the former group needing subsequent surgery in comparison to 17% of the latter group. The rate of recurrent bleeding in the mechanical therapy group was significantly lower in comparison to the injection therapy group (8.3% versus 33.3%, p < 0. 05)., Conclusions: Higher efficacy in terms of initial hemostasis and less recurrent bleeding was achieved by mechanical hemostatic therapy with hemoclip and band ligation compared with injection therapy. Endoscopic mechanical therapy is recommended as effective for bleeding Dieulafoy's lesions.

Radiation proctitis is a frequent acute complication encountered with pelvic irradiation. This study was aimed at establishing the optimal radiation dose for radiation-induced proctitis in rats. Female Wistar rats were used. The rectal specimens were examined morphologically at 5th and 10th day following 10-30 Gy irradiation in single fraction. With increasing dose, mucosal damage became worse, and there was a prominent reaction after > or =15 Gy. We selected 17.5 Gy as an optimal dose for radiation proctitis and examined specimens at day 1-14 and at week 4, 6, 8, and 12 after 17.5 Gy. The rectal mucosa revealed characteristic histological changes with time. An edema in lamina propria started as early as 1-2 days after irradiation and progressed into acute inflammation. On day 7 and 8, regeneration was observed with or without ulcer. Four weeks later, all regeneration processes have been completed with end result of either fibrosis or normal appearing mucosa. This study showed that the radiation injury of the rectum in rat develops in dose-dependent manner as it has reported in previous studies and suggested that 17.5 Gy in single fraction is the optimum dose to evaluate the protective effect of various medications for radiation proctitis in face of the clinical situation.

Membrane fusion intermediates induced by the glycosylphosphatidylinositol-linked ectodomain of influenza hemagglutinin (GPI-HA) were investigated by rapid freeze, freeze-substitution, thin section electron microscopy, and with simultaneous recordings of whole-cell admittance and fluorescence. Upon triggering, the previously separated membranes developed numerous hourglass shaped points of membrane contact (approximately 10-130 nm waist) when viewed by electron microscopy. Stereo pairs showed close membrane contact at peaks of complementary protrusions, arising from each membrane. With HA, there were fewer contacts, but wide fusion pores. Physiological measurements showed fast lipid dye mixing between cells after acidification, and either fusion pore formation or the lack thereof (true hemifusion). For the earliest pores, a similar conductance distribution and frequency of flickering pores were detected for both HA and GPI-HA. For GPI-HA, lipid mixing was detected prior to, during, or after pore opening, whereas for HA, lipid mixing is seen only after pore opening. Our findings are consistent with a pathway wherein conformational changes in the ectodomain of HA pull membranes towards each other to form a contact site, then hemifusion and pore formation initiate in a small percentage of these contact sites. Finally, the transmembrane domain of HA is needed to complete membrane fusion for macromolecular content mixing.

In order to observe the cytotoxicity of Acanthamoeba spp., which were isolated from contact lens containers as ethiological agents for the probable amoebic keratitis in Korea, the crystal violet staining method and LDH release assay were carried out. In the crystal violet staining method, among eight contact lens container isolates, isolate 3 (Acanthamoeba KA/LS5) showed 83.6% and 81.8% of cytotoxicity, and isolate 7 (Acanthamoeba KA/LS37) showed 28.2% and 25.1% of cytotoxicity, in 1 mg/ml and 0.5 mg/ml lysate treatments, respectively. Acanthamoeba culbertsoni and A. healyi showed 84.0% and 82.8% of cytotoxicity. Similar results were observed in A. castellanii and A. hatchetti which showed 83.6% and 75.5% of cytotoxicity. Acanthamoeba royreba and A. polyphaga showed 9.0% and 1.7% of cytotoxicity. In the LDH release assay, isolate 3 (20.4%) showed higher cytotoxicity than other isolates in 1 mg/ml lysate treatment. The results provide that at least isolate 3 has the cytotoxic effect against CHO cells and seems to be the pathogenic strain.

To determine whether trophozoites and lysates of pathogenic Acanthamoeba spp. induce apoptosis in primary-culture microglial cells, transmission electron microscopic (TEM) examinations, assessment of DNA fragmentation by agarose gel electrophoresis, and the TdT-mediated dUTP nick-end labeling assay were performed. When a trophozoite of pathogenic Acanthamoeba culbertsoni came in contact with a microglial cell, the digipodium was observed by TEM. Nuclear chromatin condensation was observed in 10% of microglial cells, while it was not revealed when they were cocultured with weakly pathogenic Acanthamoeba royreba trophozoites. DNA fragmentation in microglial cells cocultured with the A. culbertsoni lysate was detected by electrophoresis, showing DNA ladder formation, whereas it was hardly observed in microglial cells cocultured with A. royreba. DNA fragmentation of microglial cells was also confirmed by flow cytometry analysis. The fluorescence of TdT-stained apoptotic bodies became intensely visible with microglial cells cocultured with the A. culbertsoni lysate. In contrast, with microglial cells cocultured with the A. royreba lysate, only a background level of fluorescence of TdT-stained apoptotic bodies was detected. These results suggest that some rat microglial cells cocultured with pathogenic A. culbertsoni undergo cytopathic changes which show the characteristics of the apoptotic process, such as nuclear condensation and DNA fragmentation.

In order to refer to the basic information regarding the identification of isolates obtained from a contact lens container in Korea, the isoelectric focusing gel electrophoresis was employed to compare the isoenzyme band patterns among Acanthamoeba spp. including eight isolates and the simple pairwise dissimilarity analysis was carried out. For an alkaline phosphate development, isolate 7 and Acanthamoeba polyphaga showed homologous band patterns, and isolates 1, 2, and 3 showed the same patterns. For lactate dehydrogenase, similar patterns were observed in isolates 2 and 3. Isolates 3 and 5 showed homologous band patterns for malate dehydrogenase and glucose phosphate isomerase. For hexokinase, isolates 4, 7, and A, hatchetti showed the same band patterns. In others, a considerable number of interstrain polymorphisms was observed in nine isoenzyme band patterns. In Acanthamoeba group II, genetic distances among isolates 1, 2, 3, 4, and 5 ranged from 0.104 to 0.200. In comparison to A. castellanii, A. hatchetti, and A. polyphaga, genetic distances of isolates 7 and 8 were 0.254 and 0.219, respectively. In Acanthamoeba group III, including A. culbertsoni, A. healyi, and A. royreba, isolate 6 had genetic distances which ranged from 0.314 to 0.336. Finally, when comparing to the six reference Acanthamoeba, it was possible to classify isolates 1, 2, 3, 4, and 5, as genetically close-related species and as independent species group. Furthermore, isolates 6, 7 and 8 were identified as independent species as well.

Using the polymerase chain reaction, we examined the allele frequencies and heterozygosities of six (CA)n markers of the dystrophin gene in Koreans. Allele frequencies of these markers were different from those reported for Caucasians. The heterozygosity values for these markers range from 29 to 86%. With the exception of the STR50 marker, these values were lower than those of Caucasians. However, all markers except for the 3'CA marker showed PIC values over 0.5, suggesting a high degree of polymorphism. Therefore, this study will be useful in linkage analysis for Duchenne and Becker muscular dystrophy families in the Korean population.

Dihydropyrimidine dehydrogenase (DPD) is the major metabolic enzyme in the catabolism of 5-fluorouracil, and the activity in normal tissues shows a wide variation among individuals. Recent studies demonstrate the relevance of DPD in the pharmacokinetics, toxicity, and antitumor efficacy of 5-fluorouracil. We investigated the DPD activity in peripheral blood mononuclear cell form 114 healthy subjects in Korea. The DPD activities in healthy volunteers were shown to follow a unimodal distribution. The mean of the activity was 0.28 +/- 0.16 nmol/min/mg protein. A wide (13-fold) intersubject variability was observed (range, 0.06-0.80 nmol/min/mg protein), and, on average, DPD activity in women (0.26 +/- 0.14) was 13% lower than in men (0.30 +/- 0.16). These findings indicate that the activity of DPD in this study was higher than in reports of research with French and white American populations.

Influenza hemagglutinin (HA) and GP64 of the baculovirus Autographa californica multicapsid nuclear polyhedrosis virus induce strikingly different initial fusion pores when mediating fusion between host cells that express these fusion proteins and target cells (Plonsky and Zimmerberg, 1996; Spruce et al., 1989, 1991; Zimmerberg et al., 1994). However, in these experiments, variations in host and target membranes confounded the analysis of the role that major components of the fusion reaction play in determining initial pore characteristics. To determine the contribution of the target cell plasma membrane to the fusion pore phenotype, we studied GP64-induced fusion of stably transfected cells (Sf9(Op1D)) to either red blood cells (RBCs) or Sf9 cells. Initial fusion pores in Sf9(Op1D)/RBC and Sf9(Op1D)/Sf9 cell pairs exhibited the same conductance, and pore flickering was not observed in either combination of cells. This indicates that the target cell determines neither the size nor the reversibility of the initial pore. However, the target cell does influence the kinetics of pore formation. The waiting time between triggering and pore appearance was shorter for Sf9(Op1D)/RBC fusion than for Sf9(Op1D)/Sf9 pairs. No correlation between pore waiting time and conductance was found. This argues against a molecular model that assumes aggregation of the pore wall from a nonfixed number of components as the rate-limiting step in GP64 fusion pore formation., (Copyright 1999 Academic Press.)

Differences in the calcium sensitivity of individual secretory vesicles can explain a defining feature of calcium-regulated exocytosis, a graded response to calcium. The role of the time dependence of calcium delivery in defining the observed differences in the calcium sensitivity of sea urchin egg secretory vesicles in vitro was examined. The calcium sensitivity of individual secretory vesicles (i.e., the distribution of calcium thresholds) is invariant over a range of calcium delivery rates from faster than micromolar per millisecond to slower than micromolar per second. Any specific calcium concentration above threshold triggers subpopulations of vesicles to fuse, and the size of these subpopulations is independent of the time course required to reach that calcium concentration. All evidence supports the hypothesis that the magnitude of the free calcium is the single controlling variable that determines the fraction of vesicles that fuse, and that this fraction is established before the application of calcium. Submaximal responses to calcium cannot be attributed to alterations in the calcium sensitivity of individual secretory vesicles arising from the temporal properties of the calcium delivery. Models that attempt to explain the cessation of fusion using changes in the distribution of calcium thresholds arising from the rate of calcium delivery and/or adaptation are not applicable to this system, and thus cannot be general.

A graded response to calcium is the defining feature of calcium-regulated exocytosis. That is, there exist calcium concentrations that elicit submaximal exocytotic responses in which only a fraction of the available population of secretory vesicles fuse. The role of calcium-dependent inactivation in defining the calcium sensitivity of sea urchin egg secretory vesicle exocytosis in vitro was examined. The cessation of fusion in the continued presence of calcium was not due to calcium-dependent inactivation. Rather, the calcium sensitivity of individual vesicles within a population of exocytotic vesicles is heterogeneous. Any specific calcium concentration above threshold triggered subpopulations of vesicles to fuse and the size of the subpopulations was dependent upon the magnitude of the calcium stimulus. The existence of multiple, stable subpopulations of vesicles is consistent with a fusion process that requires the action of an even greater number of calcium ions than the numbers suggested by models based on the assumption of a homogeneous vesicle population.

To analyze the association of Epstein-Barr virus (EBV) with gastrointestinal non-Hodgkin's lymphomas arising in immunocompetent patients, 56 consecutive cases of gastrointestinal lymphomas (B-cell: 52-cases, T-cell: 3 cases, T/NK-cell: 1 case) occurring in the stomach (33 cases), intestine (22 cases) and esophagus (1 case) were investigated for the presence of EBV using polymerase chain reaction analysis as a screening method followed by EBER-1 RNA and DNA in situ hybridization (ISH) and immunohistochemistry for the expression of latent membrane protein 1 (LMP-1). Forty-seven cases demonstrated extractable DNA and EBV DNA was detected only in 4 cases. Among the, RNA (EBER-1) and DNA ISH analysis confirmed the presence of the EBV genome in tumor cells in 3 cases (T/NK-cell lymphoma of ileum, gastric high-grade B-cell lymphoma of mucosa-associated lymphoid tissue, gastric diffuse large B-cell lymphoma). Only the T/NK cell lymphoma showed diffuse positivity of tumor cells while 2 gastric B-cell lymphomas demonstrated a scattered positive reaction and no cases expressed LMP-1. Nine cases without extractable DNA by the PCR method showed no nuclear signal by EBER-1 ISH. These findings suggest that most sporadic primary gastrointestinal lymphomas in Korea are not associated with EBV.

Granzyme B is one of the serine proteases expressed in natural killer (NK) cells and activated cytotoxic T-lymphocytes. To evaluate the usefulness of granzyme B immunoreactivity in the diagnosis of T/NK-cell lymphoma, we studied 69 cases of lymphomas occurring in the upper aerodigestive tract by paraffin-section immunohistochemistry using a commercially available monoclonal antibody to granzyme B (GrB-7). All 19 cases of T/NK-cell lymphomas defined by the expression of CD56 (NHK-1) and one or both T-cell markers (polyclonal CD3 and CD45RO) expressed granular cytoplasmic granzyme B immunoreactivity. Two out of 9 cases of T-cell lymphomas showing no CD56 expression demonstrated strong granzyme B immunoreactivity. No tumor cells among 39 cases of B-cell diffuse large cell lymphomas and 2 cases of null cell diffuse large cell lymphomas were immunoreactive for granzyme B, however a few scattered granzyme B-positive reactive small lymphoid cells were consistently observed. Based on its sensitivity in this study as well as its reactivity in routinely processed tissue sections, even without heat-induced epitope retrieval technique, monoclonal antibody to granzyme B (GrB-7) can be applied as a useful marker in the diagnosis of T/NK-cell lymphomas in conjunction with CD56.

Insertion of methyl methacrylate polymer into newly reamed bony cavities has sometimes resulted in profound hypotension, cardiac arrest, or sudden death which are more common in patients with hemodynamic instability or hypovolemia. In paralysis agitans(Parkinson's disease), dramatic worsening of the disease often occurs when another illness or trauma accompanies it. And it is possible that chronic medication with levodopa can cause the loss of ability to support blood pressure. So, it involves some risk to use methyl methacrylate in chronic levodopa-treated paralysis agitans. We present a case of paralysis agitans who demonstrated profound hypotension immediately following insertion of methyl methacrylate polymer in spite of normovolemia and proper anesthetic management.

The 1.0 kb fragment carrying a gentamicin resistance determinant was subcloned from the R plasmid in clinical isolates of Serratia marcescens and its nucleotide sequence was determined. It contains an open reading frame composed of 858 nucleotides, which shows 97% homology to the aacC2 gene in nucleotide sequence. Minicells carrying the 1.0 kb insert DNA on the plasmids produced a 30 kd protein that was consistent with the expected size of the deduced amino acid sequence from the open reading frame. A search for the promoter revealed a putative promoter of which the -35 region was provided by the right end of Tn3, suggesting that the expression of the gentamicin resistance gene is dependent on the Tn3 sequence.

The replicative form of Epstein-Barr virus (EBV) DNA was studied using two lymphoblastoid cell lines, X50-7 and 6F11, which are latently infected by Epstein-Barr virus. The lytic cycle of EBV infection was induced by transfection of the cells with the BRLF1/BZLF1 coding region of the P3HR-1 defective genome. We combined two techniques to identify the productive replicative form of Epstein-Barr viral DNA in the lytic cycle-induced cells. Restriction enzyme analysis followed by Southern blot hybridization identified a significant increase in the fused fragment encompassing both ends of EBV DNA. This indicates an increase in either episomal DNA or concatameric linear DNA. Southern blot analysis of in situ lysing gels revealed that the cellular content of linear EBV DNA was also increased significantly after the initiation of the viral lytic cycle, while the amount of circular DNA remained approximately constant. We propose from these results that the source of the fused fragment encompassing both ends of EBV DNA is a concatenated linear EBV DNA molecule, and that such a concatenated molecule most likely represents a replicative form of EBV DNA in productively infected cells.

Prevalence survey of neurocysticercosis was made in a mixed epilepsy patients of Changmi Club in Korea. From February 1987 to July 1990, a total of 2,667 randomly selected patients at 27 local centers was tested for their serum levels of anti-Cysticercus antibody (IgG) by enzyme-linked immunosorbent assay. Positive rate of the antibody was 4.0% in the examined patients. The standardized antibody positive rate by provincial population was 3.1%. The rate was the highest in patients living in Cheju Do (8.4%). The patient age brackets of 0 approximately 9 years and over 50-year showed higher positive rates of the antibody. In 750 normal persons who checked up routine physical examination, the antibody positive rate was 2.1% (standardized rate was 1.8%). These seroepidemiological data disclosed for the first time the prevalence of cysticercosis in epileptic patients and in population.

Epstein-Barr virus (EBV) infectivity was measured by two different systems. One was EBV-specific antigen (EBNA) induction in EBV-negative BJAB cells, the other was immortalization of human peripheral blood B-lymphocytes upon EBV infection. Because the sensitivity of the EBNA-induction assay, which takes 2-3 days, was similar with that of B-cell immortalization, which takes 6-8 weeks, the former can be used for the rapid and convenient determination of EBV-infectivity.

Monkey skin fibroblasts were infected with simian virus 40. Cells that exhibited the viral tumor antigen were found to retain the normal morphology of actin filaments up to six days after infection. However when cells were transformed in terms of focus formation they had lost the normal actin morphology.

Extracellular freezing injury at high subzero temperatures in human polymorphonuclear cells (PMNs) was studied with a cryomicroscope, electron microscope, and functional assays (phagocytosis, microbicidal activity, and chemotaxis). There are at least four major factors in freezing injury: osmotic stress, chilling, cold shock, and dilution shock. Extracellularly frozen PMNs lose functions when cooled to -2 degrees C without a cryoprotectant. Cells lose volume on freezing to the same degree as in hypertonic exposure. PMNs have a minimum volume to which they can shrink without injury. Greater dehydration produces irreversible injury to cellular functions, and cells eventually collapse under high osmotic stress. Chilling sensitivity is seen in slowly chilled, supercooled PMNs below -5 degrees C; at -7 degrees C, functions are lost in 1 h. This injury can be prevented by the addition of Me2SO but not glycerol. Me2SO does not, however, prevent cold shock (injury due to rapid cooling), which is seen during cooling at 10 degrees C/min to -14 degrees C, but not during slow cooling at 0.5 degrees C/min. One of the problems of using glycerol as a cryoprotectant stems from the high sensitivity of PMNs to dilution shock during the dilution or removal of glycerol.

A set of lambda phages containing overlapping fragments of Epstein-Barr virus (EBV) defective DNA has been cloned from P3HR-1-superinfected Raji cells. Mapping data obtained using these cloned DNA fragments confirmed the structure of P3HR-1 defective DNA previously deduced directly from virion DNA (M.-S. Cho, G. W. Bornkamm, and H. zur Hausen, 1984, J. Virol., in press). The ability of the cloned defective DNA fragments to induce EBV antigens in transfected baby hamster kidney (BHK) cells was tested using indirect immunofluorescence assays. Up to 5% of those cells receiving a defective DNA fragment BamHI-W'C' transiently expressed a de novo nuclear antigen which was identified as being a component of the EAD complex by its reactivity with characterized EBV-positive human sera. A 20-kb clone of P3HR-1 defective DNA (EcoRI-C1) was found to induce the synthesis of a component of the VCA complex. One percent of cells transfected with this clone showed cytoplasmic fluorescence when tested with either VCA+ human sera or EBV anti-VCA monoclonal antibody. Subcloning of the EcoRI-C1 fragment localized the VCA gene to a 4.1-kb segment which maps within the BamHI-A fragment of the standard genome. This segment contains a single large open reading frame of 2.6 kb (B. Barrell, A. Bankier, R. Baer, P. Biggin, P. Deininger, P. Farrell, T. Gibson, G. Hatfull, G. Hudson, S. Stachwell, and C. Sequin, 1984, Nature (London), in press). None of the defective DNA clones were capable of inducing EBV-specific nuclear antigens (EBNAs) which is consistent with the absence of the known EBNA coding regions from the defective genome.

Superinfection of lymphoblastoid cells of EBV non-producer lines with non-transforming P3HR-1 EBV leads to the rescue of transforming virus. At least part of the recovered molecules represent recombinant DNA between superinfecting P3HR-1 EBV and resident viral genomes (Fresen et al., 1979, 1980). With high titer stocks of superinfecting P3HR-1 EBV, viral particles with early antigen (EA)-inducing properties can be rescued, indicating that under these conditions of infection input viral genomes may become replicated. Sequential blot analysis with 32P-P3HR1-EBV DNA of intracellular viral DNA synthesized following infection reveals a multiplicity-dependent pattern. High particle inputs lead to preferential synthesis of certain fragments, independent of infection of either EBV genome-free or EBV genome carrier cells. This accumulation of specific viral sequences indicates defective replication of P3HR-1 EBV DNA.

The NC37-R1 cell line, established after transformation of human cord blood lymphocytes with Epstein-Barr virus (EBV) recovered from P3HR-1 superinfected NC37 cells, spontaneously produces viral particles with transforming but without early antigen-inducing properties. Progeny virus of NC37-R1 has retained its biological characteristics of spontaneous virus release and transformation up to four cycles of transformation at present. Analysis of purified NC37-R1 virion DNA, after cleavage with restriction endonuclease Hind III and comparison of its fragments with P3HR-1 EBV as well as intracellular NC37 viral DNA using the blot hybridization technique, suggests that NC37-R1 originates from a recombination between superinfecting P3HR-1 and endogenous NC37 EBV DNA.